BIOLOGY 204/205 Advanced Genetics Laboratory

TABLE OF CONTENTS

Introduction……………………………………………………………………….p. 2

MODULE 1: Recombinant DNA……………………………………………….p. 14

MODULE 2: Gene Expression…………….…………..……………………….p. 28

MODULE 3: Detection of the ALU Insertion; DNA Fingerprinting ……… p. 40MODULE 4: Proteomics……………………………………………….………. p. 45MODULE 5: RNA Interference…………………………………………………. p. 53Appendix A: Solutions Guide……………..…………………………………….

p. 63Appendix B: Sterile Technique…………………………………………………..p. 78 Appendix C: Spread Plate Technique………………………………………...

p. 78Appendix D: Pipette Use……………………..……………………………......

p. 79Appendix E: Pipette Exercises………………………….……………………......

p. 81Appendix F: GST Plasmid Map……………………………...…………….......

p. 82Appendix G: DNA/Protein Markers……………………………………...…..

p. 83Appendix H: Streak Plate Method …………………..………………………... p. 84Appendix I: PCR Reagents and Conditions for 1.17………………………… p. 85Appendix J: Pierce Protein Assay for Module 2 ……………………...……..

p. 86Appendix K: Protein Gel Set up………………………………………………. p. 87Appendix L: Pierce Protein Assay for Module 4 …………………………….. p.88

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BIOLOGY 204/5Advanced Genetics Laboratory I and II

--- Introduction ---

Module 1 Recombinant DNA/Bacterial Transformation

This module gives you some of the experience you would receive if you were to sub-clone a gene as a part of your research. That is, once you transform a bacterial line with the plasmid that you isolate, you will need to demonstrate that you have made the transfer of the correct gene.

Goals:1. To purify a plasmid and transform E. coli with the plasmid.2. To demonstrate that the transformants carry the plasmid by

characterizing the transformants’ phenotypes. 3. Analyzing the size of the DNA plasmid in a cracking gel. 4. Hybridization with the original plasmid in a Southern blot.5. Amplify the gene inserted into the plasmid by PCR.6. Sequence part of the plasmid.

Module 2 Gene Expression

This module allows you to determine whether a cell is expressing a gene of interest, either GFP or GST. You will run the bacterial lysate on gels, stain with Coomassie blue to look for a protein of the correct size and perform a Western blot to determine whether the protein of interest was expressed.

Goals:1. Confirm, using PCR, that the plasmid from the transformed

bacteria (from module 1) has the GST gene.

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2. Show that bacteria with the gene for GST in the expression plasmid are expressing GST using gel electrophoresis of the bacterial lysate.

3. Show that GST is expressed by the bacteria using a Western blot of the lysate.

Module 3 Human Genetics: Detection of the ALU Insertion; DNA Fingerprinting

This module allows you to carry out processes and procedures that a forensic scientist might carry out.

Goals:1. Isolation of your cheek cell DNA for polymerase chain reaction

amplification.2. Analysis of PCR products by agarose gel electrophoresis for the

Alu insertions in chromosome 8 and a polymorphism called a variable number tandem repeats (VNTR) D1S80 from the noncoding region of chromosome 1.

3. Use the techniques you learned to help solve a “crime” by comparing DNA “found” as evidence, against the DNA of the “victim” and the “suspects.”

Module 4 Proteomics

In this module you will compare the proteins found in wild type and mutant paramecia cilia. You will run a one dimensional polyacrylamide gel, cut out bands of interest, and compare the proteins present in those bands using mass spec.

Goals:1. Conduct a Pierce Protein Assay to determine the concentration

of cilia proteins in samples provided to you.2. Using SDS-PAGE, run a one dimensional gel and stain with

coomassie blue to visualize differences in protein content of cilia types.

3. Cut out bands of interest from gel and prepare samples for mass spectrometry by trypsinizing proteins

4. Analyze results from mass spec

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Module 5 RNA Interference

In this module, a specific target gene product of Paramecium tetraurelia is depleted using an RNAi feeding method. You will isolate RNA from the paramecia and determine if there has been down regulation of the gene product in the RNAi treated population compared to the control.

Goals:1. Isolate RNA from harvested paramecia cells2. Synthesize cDNA from the mRNA collected3. Determine the level of target endogenous mRNA by semi-

quantitative Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR)

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Biology 204/205 Advanced Genetics Laboratory Grading Policy

Biology 204 and Bio 205 are four credit courses. You will complete Modules 1 & 2 and a grant writing exercise during the fall semester for Bio 204. For Bio 205 in the spring semester, you will complete two modules (Modules 3, 4 if you have already completed 1 and 2) and Module 5.

The format of the course is a short introduction and two formal meeting times per week. The emphasis of the course is on experimental design, techniques, data gathering and analysis. Work at the bench is given priority over work in a lecture setting. The modules are designed to approach real situations in ongoing research projects. Therefore, the modules are not necessarily designed to be finished in three hours. A few labs will run long, taking 4-5 hours to finish. A few labs will be relatively short. Students will sometimes need to return on an alternate day, usually at their own convenience, to perform a short manipulation. Sometimes an experiment does not work and it has to be repeated. Coming to class well prepared and following directions carefully will cut down on potential mistakes!

Grading:Your grade will be based on the following components, each with approximately equal weight:

1. Performance in laboratory2. Discussion of experiments in class3. Notebook (these will be checked weekly) 4. Laboratory report 5. Grant proposal (Bio 204) or Lab meeting presentations (Bio 205)6. Final oral exam covering both lecture and laboratory material

Supplies:You will need the following:

Laboratory manual Bound laboratory notebook UV safety glasses Sharpie permanent marker Transparent tape

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Note: It is important to read over the procedures in the laboratory manual and the corresponding information in the Molecular Biology Laboratory Atlas before coming to class. Be prepared to start work after an introduction by the instructor or TA. Check the laboratory calendar so that you know when each module will be done.

Laboratory Notebook Guidelines

Bound notebook; no loose-leaf Record in blue or black ink Number all pages Date all entries Name, course number and email address should be on front

cover Reserve 3 pages at the beginning for the table of contents; keep

up to date Mistakes should be crossed out with a single line through the

entry then initialed Do not skip pages, do not rip pages out Unused portions of a page should have a diagonal line drawn

through the blank portion Each experiment should begin on a new page All data, calculations and graphs should be entered directly into

the notebook Neat, orderly, complete

Your notebook should provide enough detail so that another Advanced Genetics student could pick it up and repeat your procedure by following your entries. You should include all of the following information: What was done and why, who suggested it, who did it and when it was done, what results were obtained and what conclusions were drawn.

NOTE: NOTEBOOKS WILL BE CHECKED WEEKLY AND GRADED ACCORDINGLY!

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Laboratory Report Guidelines

You will be asked to write a formal report of one of the results from one of the lab modules. You will prepare this report as you would prepare a manuscript for publication, with introduction, methods, results, and discussion sections. To aid your preparation of this report, you should go to the library early in the semester and find a short article from Genetics from the last 5 years (download a pdf version or photocopy from a paper journal, the library has both formats). The format in Genetics is appropriate for your report. Below is a description of the content and length of each section.

The report in its entirety should not exceed 10 pages in length. It should be printed double-spaced, with no less than 1-inch margins. It must be in 12-point size in a common font. Each section except the introduction should be started by its section name, in bold type. At the head of the report, you should provide a title that indicates which exercise you are writing about and your name. Whenever possible, you should strive to write succinctly and in the active voice.

Abstract: 250 words summarizing the experiment.

Introduction: The introduction provides an overview of what the report is about, including why the exercise was done (the goal of the exercise) and an explicit statement of the hypothesis or hypotheses being tested. Background information about the biology underlying the exercise should be included in the introduction. Recommended length: 1.5 pages.

Methods: The methods section must be detailed enough to allow the reader to repeat the exercise. You do not need to repeat the detailed description of the protocols in the laboratory manual, but you should refer to the methods in the manual (Format: Laboratory manual Page x-y) at the appropriate points. Recommended length: 3 pages.

Results: The results section reports upon what happened during the exercise. You must include photocopies of the final gels and provide in tabular form other measurements and data you collected. Each figure should have a brief descriptive caption, and each table should have a title. However, it is not sufficient to simply insert these figures and tables. You must interpret your results in the text of the section,

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with references to the appropriate figure or table (Format: Fig. 1, Table 2A). Recommended length: 2 pages.

Discussion: In the discussion, you should briefly re-introduce the main goal or hypothesis presented in the introduction, and then describe how your results are related to the goal or hypothesis. In subsequent paragraphs, you should discuss any failures to obtain results, and describe what you believe happened and what you would do differently to correct each problem. This is your opportunity to show how well you understand the molecular processes underlying the protocols! Recommended length: 2 pages.

Grant Writing Guidelines

The grant proposal must be based upon a novel concept that could be explored within the technological and financial limitations of our laboratory, using model organisms only. These limitations will aid you in narrowing your choice of topic.

A. Topic: The topic chosen should be novel, which means that no one else has worked on this same exact problem before. However it should also be one that can realistically attacked in the context of this laboratory. You should be able to put this topic into a broader context; Why is this an interesting problem? What has already been done with this problem in the past?

1. From this topic you must develop a testable hypothesis. This means you can develop an experiment that will result in data that leads you to clearly be able to reject or accept the hypothesis.

2. The experiment(s) will utilize techniques that you can do within this laboratory or with our collaborators.

3. If you are currently working in a laboratory outside of this class the topic you choose MUST be independent of that laboratory. You are not permitted to work on the same model organism or a topic that is related to your outside research.

4. You are permitted, indeed encouraged, to discuss possible topics with classmates, friends, family and other faculty.

B. Model Organisms: Your experiments must be limited to classic model organisms from molecular genetics that are readily available, easily

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maintained and for which we have appropriate technologies to care for and manipulate. Examples include: Drosophila, bacteria, Paramecium, C. elegans, yeast, small plants like grasses. You cannot work with organisms that are difficult and expensive to maintain (i.e. mice or other small rodents, fish, large vertebrates, humans, etc.)

C. Grant Format: Below is the format that your grant must follow. All text must be double spaced 12 point type with 1 inch margins. Make sure your grant contains all of the information within the guidelines given:

1. Cover Page - Fill out the cover page provided completely

2. Table of Contents - Page two is a table of contents. Provide the page number of each category. Number pages consecutively at the bottom right of each page throughout the application (including the cover page as page one).

3. Biographical sketch - A one page biographical sketch of the Principle Investigator (PI; this is you!). This contains your name, your date of birth, your education history, your previous relevant employment and a brief description of your prior experience that allows the reviewer to recognize you are capable of doing the proposed research.

4. Research Plan - This section should be 10 pages total. It should contain sufficient information needed to evaluate the project, independent of any other documentation. Be specific and informative, avoid redundancies. All tables, graphs, figures, diagrams and charts must be included within the 10 page limit. The following questions must be addressed in your research plan:

a. What topic will you be trying to address?

b. Why is this research important?

c. What has already been done in this field/topic?

d. What exact experiments will you be performing?

e. What are your expected results? (This is to clearly show your hypothesis. What results will allow you to accept your hypothesis? What results will cause you to reject your hypothesis?)

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5. Literature Cited - All references cited in the proposal (of which there should be many) must be listed alphabetically by first author. They must include all authors, year of publication, complete title of article, journal name (no abbreviations or websites), volume and page numbers.

6. Appendix - In an appendix not to exceed 4 pages you may provide detailed descriptions of your protocols, with all appropriate references in your citations*. Your descriptions of the protocols should be as detailed as those provided in the laboratory manual.

*Protocols: If you are using protocols in the laboratory manual you may cite the manual itself as a reference. If, however, you propose other protocols you find and cite the original reference that describes the protocol.

Use the following format and page distribution for section 4 - Research Plan:

a. Specific Aims: Clearly state your hypothesis and then list the Specific Aims of your research to test the hypothesis. Specific Aims should organize the experiments that you will do to test the hypothesis. (See example below.) List the broad, long term objectives (goals) of the research and then present the proposed project's relationship to these goals. This section should not exceed one page.

b. Background Information: Summarize the major research that has been done on this topic leading to your proposal. Critically evaluate existing knowledge, and specifically identify the gaps. State where your research will fit in with what has been done previously, what new information does your research hope to discover? This section should not exceed two pages.

c. Research Design and Methods: Describe the overall research design and the specific procedures to be used. Include how data will be collected, analyzed and interpreted. Discuss the potential difficulties and limitations of the proposed procedures and alternative approaches to achieve the aims. Also provide a timeline for the project (not to exceed six weeks.) This section should not exceed six pages.

d. Possible Results: In terms of the hypothesis you are proposing to test, state what the results are expected. Which possible results will cause you to accept your hypothesis? Which possible results will cause you to reject your hypothesis? What are the limitations

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that may not allow you to have a clear answer? This section should not exceed one page.

This information was modified from the NIH instructions for US Department of Health and Human Services Public Health Grant

http://grants.nih.gov/grants/funding/phs398/phs398.html

Example: Hypothesis is that the moon is made of blue cheese.

Specific Aim 1: Build a rocket ship to get to the moon and tools to sample cheese.

Specific Aim 2: Sample cheese on the moon and analyze it for its cheese-ness. Is it Blue or Velveeta?

Safety in the Laboratory

General Rules:1. Disinfect your bench top with a 10% bleach solution when you

arrive and when you finish lab.2. Wear gloves (lab coats, closed toe shoes and safety goggles are

also highly recommended). Avoid touching face and/or hair

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during an experiment. Chemicals on your gloves can be transferred to your face/hair.

3. Wash your hands before you leave lab.4. Do not eat, drink, smoke, chew gum or apply cosmetics while in

lab.5. Dispose of all used materials as directed.6. Keep aisles clear.7. Wipe all spills immediately. Inform lab tech and/or TA if you

spill ANYTHING.8. Dispose broken glass in the appropriate receptacle. Inform lab

tech and/or TA that you have broken glass.9. Tie back long hair.10. Try to avoid wearing baggy, loose clothing that can

interfere with your experiment and may catch on fire. 11. Extinguish burners as soon as you finish using them.12. All Chemical Safety and MSDS information is located in

the binder on the back of the door.13. If you are unsure about a procedure, just ask.

General Instructions for Recombinant DNA Work

The recombinant DNA involved in Modules 1 and 2 requires a P1 level of containment. That means you must observe the following precautions:

1. Gloves must be worn to do the lab work.

2. Hands must be washed before leaving the laboratory

3. No eating, drinking or smoking in the laboratory.

Pipetting :

1. Acquaint yourself with the various denominations of pipettors in an attempt to avoid mistakes, particularly when working under time pressure.

2. The height of the fluid in the glass pipettes is measured at the bottom of the meniscus while the pipette is being held vertically.

3. Never put a pipette back into a sterile container.

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4. Do not handle the lower part of the pipette.

Serial Dilutions: Serial dilutions allow you to dilute a sample many fold by making a series of small dilutions.

Standard Dilution Steps: Unless special circumstances demand it, the following are the only dilution steps that are used (For convenience and error avoidance in performing the accompanying arithmetic): 10, 20, 50 and 100.

10X 1:10 0.1 ml/0.9 ml 100 µL/900 µL

20X 1:20 0.1 ml/1.9 ml 50 µL/950 µL

50X 1:50 0.1 ml/4.9 ml 20 µL/980 µL

100X 1:100 0.1 ml/9.9 ml 10 µL/99 0µL

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Module 1 Recombinant DNA

Please refer to page 2 for introduction

**Note: The E.coli cell line used in this module is K12. The K12 bacteria cells are to be transformed with the GST plasmid.

1.0 Overnight (ON)Bacterial Culture (Done for you)

1. The lab tech will add 2.5 ml of cells previously grown ON to 125 ml LB amp medium (per group).

2. The cells will grow with shaking at 37C ON.

1.1 Isolation of Plasmid DNA

Three hours before class the lab tech will heat treat the RNase A. Dissolve 100 mg of pancreatic RNase A in 1 ml 10mM Tris-Cl/15mM NaCl and place in 100°C heat block for 15 minutes. Allow tubes to cool slowly to room temperature before using in Step 10. (Done for you)

HAZARDOUS CHEMICAL INFO: -Salt-Saturated Phenol is to be used only while wearing gloves under the hood. Dispose of all pipets and liquid waste containing SS Phenol in appropriate disposals. -Chloroform: Isoamyl Alcohol (24:1) is to be used only while wearing gloves under the hood. Dispose of all pipets and liquid waste containing C:IA in appropriate disposals.

1. Transfer 125ml of the overnight bacterial culture (Transformed E. coli) to a large, sterile centrifuge bottle and harvest the bacteria by centrifuging at 5000 rpm, 4ºC for 10 minutes in the Beckman J2-21.

2. Decant the supernatant broth into the waste jar.3. Resuspend the bacterial pellet in 5ml of Solution I containing

5mg/ml lysozyme.4. Transfer to a 30ml polycarbonate Oakridge centrifuge tube. Let

stand at room temperature for 5 minutes.5. Add 10ml of freshly made Solution II. Place the cap on the tube

and mix the contents by inverting the tube several times. Let stand on ice for 10 minutes.

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6. Add 8ml of ice-cold 5M potassium acetate (pH 4.8). Fill tubes only ¾ full. Screw on the cap and mix by inverting. Let stand on ice for 10 minutes.

7. Balance the tubes before centrifugation.8. Centrifuge in the Beckman J2-21; 15,000 rpm, 4ºC for 20

minutes. The genomic DNA and bacterial debris should form a tight pellet at the bottom of the tube.

9. Being very careful not to disturb the pellet, divide the supernatant in half. Transfer each half to separate 15ml glass conical tubes.

10. Add 500µL of heat treated RNase A to each tube. 11. Incubate at 37ºC for 20 minutes.12. In the chemical flow hood, add one volume of SS (salt

saturated) phenol. (Note the yellow color which helps you identify the phenol phase in the next step.) Your tubes can be no more than 2/3 full including the addition of the phenol, so divide your original solution as necessary into 3 or 4 conical tubes. SAFETY NOTE: Phenol can cause severe burns to skin and damage clothing. Gloves, safety glasses, and a lab coat should be worn when working with phenol. All manipulations should be carried out in a fume hood. A glass receptacle is available exclusively for disposing of used phenol and chloroform.

13. Vortex the conical tube and contents with lids on for 1 minute; be sure the contents are thoroughly mixed. Make sure the tops of the conical tubes are screwed on tightly to ensure that no leaking will occur. Centrifuge for 1 minute at 2800 rpm using the IEC Centra 7 Benchtop.

14. Transfer the non-colored upper, aqueous phase to a fresh conical tube. Do not take the interface which is denatured protein. In the hood, add 1 volume of chloroform: isoamyl alcohol (24:1). Vortex 1 minute and centrifuge 1 minute at 2800 rpm.

15. Transfer the upper, aqueous layer to a fresh 30ml glass tube and add 2.5 volume of cold 95% ethanol. You need to calculate how much total liquid will be in each 30ml glass tube. The tube cannot be more than 2/3 full, so you may have to use more than one 30ml glass tube. Make your calculations before adding the ethanol!

16. Mix and allow to precipitate on dry ice for 15 minutes. 17. Balance your tubes along with their rubber sleeves.18. Recover the DNA by centrifuging the tube at 4ºC in the

Beckman J2-21 at 9500 rpm for 30 minutes.19. Discard the supernatant into a waste container. The pellet

will look like a whitish residue on the side of the tube. To resuspend the pellet, use the pipette tip to scrape the sides and

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then wash the residual ethanol up onto the sides of the tube. Try to resuspend the entire pellet to increase your plasmid yield.

20. Start with one tube: wash the pellet with 1ml 70% ethanol by pipetting up and down to resuspend the pellet. Transfer resuspention to the next tube until all pellets are resuspended and pooled together. Transfer the solution into one sterile 1.5ml microfuge tube.

21. Microcentrifuge for 5 minutes at 14,000 rpm. Discard the ethanol; add 1ml more of 70% ethanol to wash the pellet and vortex for 30 seconds. Spin at 14,000 rpm for 5 minutes.

22. Discard the ethanol; dry the pellet using the SpeedVac in the basement. Give your sample to the TA/Lab tech to be properly dried.

23. Dissolve the pellet in 0.3ml TE. Aliquot 100 L to each of 3 microcentrifuge tubes (properly labeled!).

24. Store at -20ºC.

1. 2 Agarose gel to confirm isolation of the plasmid HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.-UV light is very harmful if looked at directly. When viewing your gels on the UV light box be sure to wear a protective face mask, or place the shield on top of the box before turning on light.

1. Prepare 300ml 1X TAE from 5X TAE stock.2. Dissolve 0.35g agarose in 50ml 1X TAE buffer to make a 0.7%

gel.3. Microwave on high for 1 minute.4. Swirl the flask and make sure all of the agarose is dissolved. If

not, microwave until it is.5. Place the running tray into the gel-casting tray. Add comb. 6. Cool agarose slightly; approximately 5 – 10 minutes, swirling

occasionally. Slowly pour agarose into the farthest corner from the comb in the gel casting set up. Try to avoid bubbles! If bubbles appear remove them with a pipette tip. Let cool until opaque (approximately 20 minutes).

7. While your gel is setting, thaw out one tube of your plasmid DNA on ice. Just before you are ready to load the gel, heat the λ Hind III marker for 7 minutes in the 65ºC hot block.

8. Mix 4µL of 6X DNA sample buffer with 20µL plasmid DNA on a piece of Parafilm.

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9. Once your gel is set, remove it from the casting tray. Place it in the running tray, with the comb still set. Cover the gel with 1X TAE. Gently remove the comb. Removing the comb last will ensure that your wells do not collapse.

10. Be prepared to load the get quickly—you do not want your DNA to diffuse into the running buffer.

11. Load 24µL of plasmid DNA sample and 20µL of λ Hind III marker in the wells; put the lid on the box so that the DNA will run toward the red electrode.

12. Run the gel at 100V for ~1 hour.13. Stain the gel for approximately 15 minutes in ethidium

bromide, and destain in water for 5 minutes.14. Examine the gel on the UV light box. If the ladder is not

visible or is faint, place the gel back into the stain. When you feel that your gel is properly stained, take a picture to document your results.

15. Leave the gel in destain to be discarded later.

1.3 Grow an overnight broth culture of E. coli (Done for you)

1.4 Transformation

Three hours before class the tech will take 1 ml of an ON culture and inoculate 50 ml of fresh LB broth with it. It will shake at 37C for three hours. This will produce exponentially growing cells for you to transform.

1. Divide broth culture into 2 sterile 30ml Oakridge centrifuge tubes; place tubes in ice for 30 minutes.

2. Thaw out one tube of your plasmid DNA on ice.3. Centrifuge the cultures at 4ºC in the Beckman J2-21 for 7

minutes at 5000 rpm; decant the supernatant into the collection flask provided.

4. Resuspend one pellet in 25 ml ice cold 50 mM CaCl2. Combine this resuspension solution with the second bacterial pellet; place on ice for 20 minutes. Keep CaCl2 on ice while waiting.

5. Centrifuge the cell suspension at 4ºC in the Beckman J2-21 for 7 minutes at 5000 rpm.

6. Decant the supernatant and resuspend the pellet in 3ml ice-cold 50 mM CaCl2; place on ice for 5 minutes.

7. Dispense 2 aliquots of 0.3 ml cells in ice-cold 1.5ml microfuge tubes; add 0.2 ml of transformation buffer to each tube.

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8. Add 5 µL [>2 g] plasmid DNA to one tube. The second tube will not contain plasmid DNA and will act as a control. Mix gently and leave on ice for 20 minutes.

9. Heat shock cells for 1 min in 42°C water bath.10. Plunge tubes into ice and let sit on ice for 5 minutes.11. Add 0.7 ml LB to each tube and tap gently with finger.12. Shake at 37ºC for 60 minutes.

**NOTE: during this hour incubation your TA or Lab Tech will demonstrate proper spreading and streaking procedures for plating. It is very important that you understand sterile technique when working with bacteria so you don’t contaminate your samples.

13. Plate 0.05, 0.1, and 0.3 ml of the cells with plasmid DNA onto LB amp plates. Use the spread plate technique. Let the plates dry for 5 minutes right-side up before inverting and placing in the incubator.

14. Streak (Do not use the spread plate technique) the contents of the “no DNA tube” on an LB amp plate and an LB plate. The LB amp plate will act as a negative control, while the LB plate will serve as a positive control.

15. Label plates appropriately with group number, date, type of bacteria, and any other important information, such as how much bacteria was plated.

16. Incubate the plates at 37ºC overnight (upside down); be sure to remove, wrap in Parafilm and refrigerate the plates tomorrow!

Following Day: 1.5 Selecting for bacteria that carry the plasmid___

1. Examine transformed and no DNA control plates. (There should be no colonies on the “No DNA” plate)

2. Choose 6 well isolated colonies from the transformed plates. Streak each colony on half of an LB amp plate.

3. Choose 2 well isolated colonies from the control (non-transformed) plate provided. Streak each colony on one half of an LB plate.

4. Incubate the plates overnight at 37ºC.5. Wrap the old plates in Parafilm and refrigerate.

1.6_Secondary selection of transformed bacteria_________________

1. Transfer 4 well-isolated colonies from 4 different transformed streaks and 2 control colonies into separate 1ml aliquots of sterile saline. Refrigerate the old plates.

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2. For the transformed bacteria, streak 1 loopful of saline/bacteria suspension onto ½ of an LB amp plate. Do this for each of the 4 samples.

3. For the control cells, streak 1 loopful of the saline/bacteria suspension onto ½ of an LB plate. Be sure to label plates clearly!

4. Incubate at 37°C overnight; remove and refrigerate the next day.

5. Go to 1.16

1.7 Preparing bacteria for the cracking gel (day before 1.8)

1. Using a marker, draw a line down the center of a new LB amp plate. Make a template on paper with 1.5 cm x 1.5 cm squares on each half. Place the plate over the template.

2. Using sterile tweezers, select a sterile toothpick.3. Choose 2 LB amp plates from Day 1.6 that show the best

growth. With the toothpick, select one colony from the Day 1.6 plate and “fill in” the square on the agar on the plate. Repeat for the 2nd colony using a new toothpick.

4. Repeat the procedure for the control, but use a fresh LB plate.5. Incubate at 37ºC overnight for at least 24 hrs, but less than 36

hrs.

1. 8 Next day: Cracking gel HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 300ml 1X TAE.2. Prepare 0.7% agarose gel.3. Use a sterile toothpick to scrape bacteria from the plates

prepared the day before. Add bacteria from each square to 250µL of cracking buffer (Two squares for one tube of 250µL of cracking buffer). Do this for transformed and non-transformed cells (you should have a total of 2 microcentrifuge tubes). Vortex tubes to mix well.

4. Incubate at 37ºC in the hot water bath for 25 minutes.5. Centrifuge for 15 minutes at 14,000 rpm.6. Use a toothpick to remove the bacterial debris from the bottom

of each tube. (You won’t be able to see a pellet, but when you pull it out, it will look like a blue glob).

7. Load the gel:

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8. Lane 1: 20µL Hind III marker (Heat in 65C hot block for 7 minutes before loading) Lane 2: 20µL plasmid DNA solution (10µL plasmid DNA + 4µL 6X DNA sample buffer + 6µL 1X TAE)Lane 3: Transformed supernatant Lane 4: 50µL Cracking buffer only Lane 5: Non-transformed supernatant Note: Load as much transformed and nontransformed supernatant as possible (A well formed well can hold ~50 L).

9. Run the gel for 1 hour at 100 volts.10. Stain with ethidium bromide, destain, and photograph.

Look for genomic DNA, plasmid DNA and RNA.

1.9 Labeling DNA with Biotin -Salt-Saturated Phenol is to be used only wearing gloves under the hood. Dispose of all pipets and liquid waste containing SS Phenol in appropriate disposals. -Chloroform is to be used only wearing gloves under the hood. Dispose of all pipets and liquid waste containing chloroform in appropriate disposals.

Part A: Labeling Reaction1. Remove an aliquot of Plasmid DNA from the refrigerator and

place on ice. 2. Add labeling reaction components to a 0.5ml tube (on ice):

dH2O 128µLdNTP mix 28µL1X DNase I Buffer 19.9µLDNase I 0.1µLPlasmid DNA 4µLDNA Polymerase I 20µL

3. Mix well and centrifuge for 5 seconds at 14,000 rpm.4. Allocate 50µL into 4 tubes.5. Incubate at 15°C for 2 hours in thermocycler.6. Add 5µL Stop Buffer to each tube and mix.7. Incubate tubes at 65°C for 5 minutes in thermocycler.

Part B: Purification of DNA probes1. Transfer liquid to consolidate solution from 4 tubes into one

tube.2. Add 4µL 10% SDS to tube and mix.3. Add 110µL Chloroform and 110µL SS Phenol to an empty

1.5ml microcentrifuge tube.

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4. Transfer DNA solution to chloroform phenol tube. Vortex 2 minutes and then centrifuge for 2 minutes at 14,000 rpm

5. Collect the top layer of liquid and transfer to a fresh 1.5ml tube. Discard remaining liquid into waste container.

6. Add 220µL chloroform to tube. Vortex 2 minutes and then centrifuge for 2 minutes at 14,000 rpm.

7. Collect top liquid layer and transfer to a clean tube. Discard remaining liquid into waste container.

8. Add 40µL 3M Sodium Acetate (pH 4.8) and 800µL cold 95% ethanol. Mix gently by inverting tube.

9. Store at -20°C ON (at least 6 hours)The Next Day:

10. Centrifuge for 5 minutes at 14,000 rpm.11. Carefully remove the supernatant.12. Resuspend the pellet in 1ml cold 70% ethanol. Centrifuge

for 5 minutes at 14,000 rpm. 13. Remove supernatant (ethanol). Let tube dry in cabinet for

at least 1 hour. 14. Once dry, resuspend probe in 12µL TE buffer and store at

-20° C.

1.10 Preparing for the Southern Blot (day before 1.11)

1. Using a marker, draw a line down the center of the underside of a fresh LB amp plate. Draw two 1.5 cm x 1.5 cm squares on the underside of the plate, one on each half.

2. Using a sterile toothpick, pick one isolated colony from the Day 1.6 LB amp transformed plate. “Fill in” one square on the fresh LB amp plate with one colony. Repeat for the second square making sure to use a fresh toothpick.

3. Repeat steps one and two, this time using an LB plate and the Day 1.6 non-transformed cells.

4. Incubate both plates for at least 24 hours.

1. 11 Southern Blot HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Run cracking gel (same as Day 1.8, except add 20ul of plasmid mixed with 4ul 6X DNA sample buffer to plasmid lane). Do not forget control lane!

21

2. Stain with ethidium bromide, briefly destain, and examine the gel.

3. Make sure to destain the gel for approximately 5 minutes before denaturing.

4. Photograph the gel before destaining completely—you will use this photograph later to compare to the results of your southern blot.

5. Denature gel in 0.5 M NaOH/0.8 M NaCl for 30 minutes, rocking. Decant the solution and repeat.

6. Rinse gel in dH2O for 1 minute.7. While the gel is rinsing, cut and hydrate the nitrocellulose filter

for 3 minutes in dH2O, then in 10X SSC until blot set-up is ready. Make sure to notch the corner of the nitrocellulose for orientation purposes and always wear gloves when handling the nitrocellulose. Always handle the filter with forceps, and only around the edges so as to not create blotches of background color.

8. Neutralize gel in 0.5 M Tris/1.5 M NaCl (pH 7.0) for 30 minutes, rocking. Decant the solution and repeat.

9. Rinse the gel in 10X SSC for 3 minutes, rocking.10. While the gel is neutralizing, prepare the Test Spot.

a. Take your Biotin labeled probe out of the freezer and let thaw on ice.

b. While thawing, cut a small piece of nitrocellulose and hydrate in dH20 for 3 minutes.

c. Soak nitrocellulose in 10X SSC until the probe is thawed.

d. Using 2 µL of probe, make a small dot on the filter.e. Let dry on a small Kimwipe, then wrap in plastic

wrap and store in the freezer until 1.12.11. Assembling the Southern Blot:

-First the wick (a long strip of paper towel will work) needs to be placed on the platform so that it can only touch the buffer on two sides.

-Place three squares of Whatman 3M filter paper on top of wick.

-The gel should be placed on top of the filter paper, and the nitrocellulose on top of that. **Make sure the nitrocellulose and the gel are lined up in the correct orientation so you can compare them later**

-Place three more squares of Whatman 3M filter paper on top of the nitrocellulose.

-A stack of cut paper towels at least 10 cm high should be assembled and tied together with string. This can go on top of the filter paper.

22

-Pressure should be applied to the top of the stack to enhance wicking overnight. (Your TA should demonstrate this and assist in the

assembly)12. Let Southern Blot transfer ON in 10X SSC.

1.12 Drying of Blot (Done for you)______________________________

1. Disassemble blot and rinse nitrocellulose filter in 5X SSC for 2 minutes.

2. Dry on large Kimwipe and then wrap in plastic wrap.3. Bake filter and test spot in vacuum oven at 80ºC for 2 hours.4. Store in freezer.

1.13 Hybridization of the Southern Blot

1. Denature 200L of Herring sperm DNA (2mg/mL) by boiling for 10 minutes followed by fast chilling on ice.

2. While the Herring sperm DNA is boiling, soak the nitrocellulose filter and the test spot in 2X SSC until uniformly hydrated.

3. For prehybridization of the nitrocellulose filter, add the 200µL of freshly denatured Herring sperm to the prehybridization solution. Mix and then transfer to a plastic tub. Add the nitrocellulose filter and test spot to the tub.

4. Incubate at 42ºC for 2 hours. The volume of prehybridization solution used should be 20 to 100L per cm2 of the filter, ~7.5ml.

5. For hybridization, heat-denature 5L of the probe made on 1.9 and 200 L of Herring sperm DNA by boiling for 10 minutes in a boiling water bath and then place on ice. Just before use, add to the hybridization solution.

6. Remove the prehybridization solution from the tub and add the hybridization solution to the filters (20-100 L per cm2), ~7.5 ml. The filter should be hybridized at 42ºC overnight to achieve maximal sensitivity.

Following Day: 1.14 Detection of the DNA

**All the washes in this section need to be completed while rocking.**

Decant and save the hybridization solution in an appropriate size tube. Store at 4C.

1. Wash the filter & test spot with 100ml of 2X SSC/0.1% (w/v) SDS at room temperature for 3 minutes. Decant the SSC and repeat.

23

2. Wash the filter & test spot with 100ml of 0.2X SSC/0.1 % (w/v) SDS at room temperature for 3 minutes. Decant the SSC and repeat.

3. Wash the filter & test spot in 100ml of 0.16X SSC/0.1% (w/v) SDS at 50ºC for 15 minutes. Decant the SSC and repeat.

4. Rinse the filter & test spot in 100ml of 2X SSC at room temperature for 1 minute.

5. Dry on large Kimwipe and then wrap in plastic wrap and store in refrigerator.

6. The hybridization mixture containing the biotin-labeled probe may be reused. Store the mixture at 4C for several days or at -20C for longer periods. Placing the hybridization solution in a boiling water bath and cooling on ice just prior to use should denature the probe.

1. 15 Development of Blot HAZARDOUS CHEMICAL INFO:-NBT/BCIP is highly toxic. WEAR GLOVES when handling and dispose of all liquid waste containing NBT/BCIP in the appropriate waste container.

1. Wash the filter and small test squares in Buffer 1 at room temperature for 1 minute with sufficient buffer to cover the blots. Decant Buffer 1 into the sink.

2. Incubate filters in Buffer 2 in a plastic container for 1 hour at 65ºC, rocking, with sufficient buffer to cover the blots.

3. Wash the filters in freshly made strep-avidin alkaline phosphatase (SA-AP) conjugate for 15 minutes at room temperature. (Add only enough SA-AP conjugate to cover the filters (~10ml). Use gentle agitation and occasionally pipette SA-AP over the filters.)

4. Decant and save the SA-AP in a 15ml tube. Save for step #6.Wash the filters in Buffer 1 using 20 to 40-fold greater volume than employed in step 3. Gently agitate filter for 15 minutes in Buffer 1.(if you used 7 ml diluted SA-AP conjugate in step 3, wash with at least 140-280ml Buffer 1.) Decant Buffer 1 into the sink.

5. Wash the filters for 10 minutes in Buffer 3, rocking. Decant Buffer 3 into sink.

6. Add 1ml NBT/BCIP solution to the saved SA-AP. A blue color should develop overtime. Wear gloves when working with NBT-BCIP.

24

7. Add 9 ml of NBT-BCIP solution to the filters. Allow the filters to develop for 15 minutes to 1 hour. (Do steps 6 and 7 at the same time and monitor the rate of color development. The tube of saved SA-AP acts as a positive control.)

8. DNA bands will be most evident on only one side of the filter (check your notch for correct orientation). Check your filter every 2 minutes to ensure that over-development does not occur.

9. Once bands have developed, decant the NBT-BCIP solution in the appropriate waste container and wash the filter in TE. This will terminate the color development reaction. The TE can then be decanted into the sink.

10. Let the blots dry on a large Kimwipe. Then wrap in plastic wrap and label. The lab tech will photograph and distribute the blots for your notebooks.

11. Measure the photograph of the cracking gel, and compare the relative position of the plasmid band to the results of the blot. Interpret your results.

1.16 Designing Primers 5’-------------------------------------------------------------------------------------------------------3’ 698 bp

GST

Partial DNA Sequence for GST (Read left to right, top to bottom):

5’…GTATTCATGTCCCCTATACTAGGTTATTGAAAATTAAGGGCCTTGT

310GCAACCCACTCGACTTCTTTTGA……….ATCCTCCAAAATCGGATCTGGT

960TCCGCGTGGATCCCCGGGAATTCATCGTGACTGACTA………….…………..3’

The glutathione S-transferase protein consists of 232 amino acids. The sequence—using the one-letter abbreviation for each amino acid—is shown below.

25

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPGIHRD

Using this information, design the primers to amplify the GST gene. Once you have designed the primers, fill out the oligonucleotide request form. The primers will then be made on a DNA synthesizer.

1. 17 PCR

1. Set up 7 - 0.5 ml PCR reaction tubes according to the PCR chart in Appendix I. Read the chart carefully and make sure you add the correct amounts of reagents. PCR is a very sensitive reaction and adding the incorrect amounts of reagents may cause poor results. Appropriately label your tubes with your group number and tube number!

2. For Sample 4, pick up three individual colonies from your transformed plate with a sterile toothpick and place into a 1.5 ml microfuge tube filled with 50 µL of sterile dH20. Take 3µL of this bacterial solution and use as your “template DNA”. Do the same for Sample 5 using non-transformed bacteria.

3. Before mixing the reactants, you must calculate how much water must be added to make a total of 50L (including the Taq Polymerase). This is necessary because the amount of template DNA that you add might differ from tube to tube.

4. If you add too much DNA, nonspecific amplification may occur—ask your TA how much DNA to add based on the approximate concentration of your plasmid samples.

5. Add all reactants, except the Taq, while the tubes are on ice.6. Lastly add the Taq polymerase.7. Once all reactants are added to the tubes, spin them briefly to

bring all the liquid to the bottom of the tube. Note: Only spin tubes briefly (5 sec.), 0.5 ml tubes are thin-walled and can crack if microfuged for too long.

8. Keep the tubes on ice until the entire class is ready to load the thermocycler.

9. The thermocycler will run for approximately 3hrs. After the 3hr. period is over, the thermocycler will stay at a constant 4C until the tubes can be placed in the refrigerator by the lab technician or TA. This will ensure that the PCR products will not degrade.

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1.18 Examining the PCR product HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 300 ml 1X TAE.2. Prepare a 2% agarose gel. Note: The 2% agarose solution

will solidify quickly! Pour gel while still relatively hot. 3. Remove 20µL of PCR product from each tube; add to 4 µL of

sample buffer. Store the remaining PCR product at 4ºC. 4. Once your gel is set, remove the comb and place the gel in the

running box. Cover the gel with 1X TAE buffer. 5. Load your DNA samples and 20µL of 100bp ladder into the gel. 6. Run gel at 100 volts for 1 hour.7. Once the electrophoresis is complete, stain your gel for ~15

minutes in ethidium bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.

8. Destain, examine, and photograph gel.

1.19 Searches of the sequence using BLAST ( B asic L ocal A lignment S earch T ool) The plasmid DNA has been sequenced. You will receive a printout of the results. You will analyze this information using a computer program called BLAST.

To access the program, go to http://www.ncbi.nlm.nih.gov/BLAST

27

Module 2Gene Expression

Introduction In the following series of experiments, you will not be using the transformed cells you created in Module 1. K12 cells do not perform as well in expression experiments, so BL21 cells will be used in Module 2. BL21 cells express the GST protein much clearer. With these cells, you will induce the expression of the Glutathione-S-transferase (GST) and run a SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) to show that the protein was expressed. You will also perform a Western blot and use antibodies to confirm the presence of the GST.

2.1: Preparation of bacteria for SDS-PAGE (day before 2.2)

1. Using a sterile toothpick, select one colony from a plate of freshly transformed BL21+GST cells and add to 10ml of LB-amp broth. Shake tube at 37C ON.

2. Add one control (non-transformed) BL21 colony to 10ml LB broth. Shake tube at 37C ON.

2.2: SDS-PAGE preparation and Pierce Protein Assay

HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. -N-Butanol is flammable. Use caution.

Four hours before class …inoculate bacterial cultures (Done for you):

1. Inoculate 2 tubes of 10ml of LB amp broth with 500L of transformed ON bacterial culture each.

28

2. Inoculate 2 tubes of 10ml of LB broth with 500L control (non-transformed) ON bacterial culture each.

3. Shake at 37C for 1 hour.4. To ONE of the LB amp cultures and ONE of the LB cultures, add

300L of 100mM IPTG each. Label tubes to indicate the addition of IPTG.

5. Shake at 37C for an additional 3 hours.

At lab time:6. Obtain a large and small glass plate; wipe with methanol and a

Kimwipe until you hear a “squeaky” noise. Handle glass plates at edges. Wear gloves!

7. Assemble the gel casting apparatus (See diagram in Appendix K). Assemble on a flat surface and then tighten screws.

8. Insert the comb and use a Sharpie to draw a line across the glass 0.7cm below the comb. Once the line is drawn, remove the comb.

9. Test to see if the apparatus is leak-proof. Squirt some water in between the glass plates and look for leaks. If leaks occur, a tighter seal must be achieved. Placing several layers of paper towels on top of the rubber seal can do this. Make sure to remove the water before pouring the gel. Use a Kimwipe to remove water droplets from between the plates of glass.

Before you pour your gel, have the TA or lab tech check your apparatus!

10. Prepare the resolving gel in a 15 ml tube according to the following directions. This recipe makes enough for two gels, so work in groups of 4. DO NOT ADD THE FRESHLY MADE AMMONIUM PERSULFATE UNTIL YOU ARE READY TO POUR THE GEL

Sterile dH2O 3.29 ml 4X resolving Buffer pH 8.9 2.60 ml

30% Acrylamide stock 4.00 ml TEMED 10 L

Last: Fresh 10% Ammonium persulfate 100 L

11. Gently swirl the solutions to mix WELL. 12. Using a Pasteur pipette, pour the gel by allowing the

acrylamide solution to run down along the side of the spacer. Add the acrylamide solution until it is just barely above your Sharpie line. Try to avoid making bubbles.

13. Overlay the acrylamide with N-Butanol to aid in the polymerization. Do this by gently adding the N-Butanol with a glass Pasteur pipette. You will be able to see a distinct line between the N-Butanol and the 4X Resolving solution.

29

14. Allow the gel to polymerize for AT LEAST 30 minutes (Any extra acrylamide mix in your tube will be a good gauge for polymerization. Make sure the cap is on).

15. While your gel is polymerizing, you can prepare your samples and standards for the Pierce Protein Assay if you have not done so already. Go to next section for instructions.

16. Once your gel has polymerized, pour off the N-Butanol into the sink and rinse with dH2O. Add a layer of dH2O on top of your gel using a Pasteur pipette.

17. Wrap the gel/casting apparatus in a damp paper towel and then plastic wrap. Label appropriately. Store in the cold room.

Prepare the proteins for the gel and protein assay: 1. Obtain 2 ml of each type of culture (Transformed, Transformed

+ IPTG, Untransformed, and Untransformed + IPTG). Split each culture into two 1.5 ml microcentrifuge tubes. You will have a total of 8 tubes (2 x Transformed -IPTG, 2 x Transformed +IPTG, 2 x Untransformed-IPTG, 2 x Untransformed +IPTG).

2. Spin all tubes in the microcentrifuge for 1 minute.3. Decant the supernatant from each tube. 4. Resuspend one of each kind of pellet (i.e. transformed,

transformed plus IPTG , -control, and +control) in 100L lysis buffer.

5. Transfer this solution to its complementary tube. Do this for each type of pellet. You will now have a total of 4 tubes.

6. Sonicate each sample in the cold room, 3 times at 10-second intervals. Sonicating breaks open the cells by sound waves. EAR PROTECTION REQUIRED!When sonicating, avoid touching the sides of the tube with the tip of the sonicator. The sample may become frothy; try to keep the sample from coming out of the tube. Turn off the sonicator and rinse the tip with dH2O in between samples and wipe with a Kimwipe.

7. Record the approximate total volume of each sample.8. Take 15 L samples from each sonicate; keep on ice for protein

assay.9. Freeze the remaining samples at -20C. Label appropriately!

Pierce Protein Assay (See directions in Appendix J)

Use the chart provided to develop a standard curve using BSA standards and to determine protein concentration.

1. Dilute protein sample: Make 10X and 20X dilutions for each protein sample. For example, to make a 20X dilution, add 5L

30

of your sample to 95L of sterile dH2O. For a 10X dilution add 10L of your sample to 90L of sterile dH2O.

2. Make dye solution: Use Solutions A and B from the Pierce Protein Assay Kit. They should be mixed 50:1…but make up only the amount you will need. Mix the dye in a 50 ml tube.

3. Add BSA and dH20 according to the directions in Appendix J.4. Add 2ml of the dye to each one of your samples and standards.5. Incubate at 37C for 30 minutes. 6. Get OD values for standards and samples: TA will assist in

the operation of the spec. 7. Place your standard into a clean cuvette.

To clean the cuvette, rinse with dH2O. Make sure to dry the outside of the cuvette with a Kimwipe. Handle the cuvette only on the frosted sides.

8. Read OD at 562nm. 9. Repeat for each standard and sample, including duplicates.

If only using 1 cuvette, make sure to rinse with dH2O between each standard.

10. Once the standards are complete, read your samples. You should blank the instrument with dH2O and dye. If using only 1 cuvette, make sure to rinse with dH2O between each sample.

11. Find the average of each standard and sample duplicates.12. Establish a standard curve using the OD values

obtained with your BSA standards: graph Concentration (x-axis) vs. OD (y-axis) on graph paper. Using this graph, calculate the protein concentrations in your three samples.

13. Use Excel to plot your data on a second chart (This is homework). Make sure to paste your Excel chart in your notebook properly labeled.

2. 3 SDS-PAGE HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the APS) in the appropriate waste container.

31

-Coomassie Blue Stain and Coomassie Blue Destain are highly flammable and irritating to the skin. WEAR GLOVES when handling and dispose of in appropriate waste container.

1. Remove the resolving gel from the cold room and pour a 4% stacking gel.

2. Mix the following components in a 15 ml tube (makes enough for 2 gels).

3. Sterile dH2O 6.10 ml 4X stacking buffer pH 6.8 2.50 ml

30% Acrylamide stock 1.30 ml TEMED 10 L

Last: Fresh 10% Ammonium persulfate 50 L

4. Before adding the ammonium persulfate, pour the dH2O off the resolving gel and dry with a Kimwipe.

5. Add the ammonium persulfate to your tube. Mix gently.6. Pour the stacking gel as you did the resolving gel all the way to

the top of the small glass plate. If it overflows when inserting the comb this is okay. Clean the comb thoroughly with methanol before inserting.

7. Being careful to avoid making air bubbles, insert the clean comb until there is no air between the wells. This is VERY IMPORTANT; the stacking gel will not polymerize if the comb is not clean, or if there is air between the wells. Ask your lab tech to double check your set up.

8. Allow 30 minutes for the gel to polymerize. Thaw your protein samples on ice while waiting.

9. Once the gels are set, remove them from the casting stand and assemble in the gel box. Note: Do not remove the comb yet.

10. Add ~115ml of PAGE Running buffer to the upper chamber. The buffer level should be half way between the big and small glass plate.

11. Add enough PAGE Running buffer to the lower chamber to cover the bottom half of the gels.

12. Carefully remove the comb.

Gel Set Up1. Calculate the volumes of sample, sample buffer, and water

needed for each tube (make 60µl total so that even if some evaporates during boiling, there will still be 40µl left):

-Each tube needs 60µg of protein total. Using the concentrations you calculated off of your standard curve, calculate how many microliters equals 60µg.

32

-Each tube needs 1X SDS sample buffer. You are given 6X SDS sample buffer. Calculate how much 6X SDS sample buffer is needed so that the final concentration is 1X.-Each tube needs a total volume of 60µl. Figure out how much water should be added to each so that the total volume is 60µl.

2. Add the calculated volumes of water, protein, and 6X sample buffer to a 1.5ml tube. Place the remaining protein samples in the freezer.

3. Boil samples for 5 minutes right before you are ready to load. After boiling, keep samples on ice while loading gel.

4. Load you samples into the gel in the following order (use gel loading tips): Lane 1, 6: Prestained protein marker (20 L per lane) Lanes 2, 7: TransformedLanes 3, 8: Transformed plus IPTG Lanes 4, 9: Control (non- transformed)Lanes 5, 10: Control (non- transformed) plus IPTG

5. Run the gel at 50mA for 1-1 ½ hours.6. Remove gel carefully from the gel apparatus. Use a razor blade

to cut the stacking gel portion away. Dispose of the stacking gel in the appropriate waste container.

7. Put the gel into a plastic container and cover with Coomassie stain. Microwave on low for 1 minute. Discard stain in appropriate waste container.

8. Transfer gel to destain. Wash and discard destain in correct waste container.

9. Rinse gel with destain again and decant destain into correct waste container.

10. Submerge the gel in more destain. Rock gently overnight.

2.4 Dry Gel: Done for you

1. Discard destain in appropriate waste container.2. Rinse gel in dH2O. Discard into appropriate waste container.3. Submerge your gel in gel drying buffer and place back on the

shaker ON.4. Photograph and appropriately dry gels using cellophane. 5. Make sure to observe and record gel appropriately.

2. 5 Pouring a Resolving Gel for SDS-PAGE and Western Blot HAZARDOUS CHEMICAL INFO:

33

-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the APS) in the appropriate waste container.

1. Pour a 12% polyacrylamide resolving gel:2. Obtain a large and small glass plate; wipe with methanol and a

Kimwipe until you hear a “squeaky” noise. Handle glass plates at edges. Wear gloves!

3. Assemble the gel casting apparatus (See diagram in Appendix K). Assemble on a flat surface and then tighten screws.

4. Insert the comb and use a Sharpie to draw a line across the glass 0.7cm below the comb. Once the line is drawn, remove the comb.

5. Test to see if the apparatus is leak-proof. Squirt some water in between the glass plates and look for leaks. If leaks occur, a tighter seal must be achieved. Placing several layers of paper towels on top of the rubber seal can do this. Make sure to remove the water before pouring the gel. Use a Kimwipe to remove water droplets from between the plates of glass.

Before you pour your gel, have the TA or lab tech check your apparatus!

6. Prepare the resolving gel in a 15 ml tube according to the following directions. This recipe makes enough for two gels, so work in groups of 4. DO NOT ADD THE FRESHLY MADE AMMONIUM PERSULFATE UNTIL YOU ARE READY TO POUR THE GEL

Sterile dH2O 3.29 ml 4X resolving Buffer pH 8.9 2.60 ml

30% Acrylamide stock 4.00 ml TEMED 10 L

Last: Fresh 10% Ammonium persulfate 100 L

7. Gently swirl the solutions to mix WELL. 8. Using a Pasteur pipette, pour the gel by allowing the acrylamide

solution to run down along the side of the spacer. Add the acrylamide solution until it is just barely above your Sharpie line. Try to avoid making bubbles.

34

9. Overlay the acrylamide with N-Butanol to aid in the polymerization. Do this by gently adding the N-Butanol with a glass Pasteur pipette. You will be able to see a distinct line between the N-Butanol and the 4X Resolving solution.

10. Allow the gel to polymerize for AT LEAST 30 minutes (Any extra acrylamide mix in your tube will be a good gauge for polymerization. Make sure the cap is on).

11. Once your gel has polymerized, pour off the N-Butanol into the sink and rinse with dH2O. Add a layer of dH2O on top of your gel using a Pasteur pipette.

12. Wrap the gel/casting apparatus in a damp paper towel and then plastic wrap. Label appropriately. Store in the cold room.

2. 6 SDS-PAGE gel and Western Blot

HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the APS) in the appropriate waste container. -Western Blot Transfer Buffer is flammable. Wear gloves and use caution when handling.

1. Remove the resolving gel from the cold room and pour a 4% stacking gel.

2. Mix the following components in a 15ml tube (makes enough for 2 gels). Sterile dH2O 6.10 ml 4X stacking buffer pH 6.8 2.50 ml 30% Acrylamide stock 1.30 ml TEMED 10 L

Last: Fresh 10% Ammonium persulfate 50 L

13. Before adding the ammonium persulfate, pour the dH2O off the resolving gel and dry with a Kimwipe.

14. Add the ammonium persulfate to your tube. Mix gently.

35

15. Pour the stacking gel as you did the resolving gel all the way to the top of the small glass plate. If it overflows when inserting the comb this is okay. Clean the comb thoroughly with methanol before inserting.

16. Being careful to avoid making air bubbles, insert the clean comb until there is no air between the wells. This is VERY IMPORTANT; the stacking gel will not polymerize if the comb is not clean, or if there is air between the wells. Ask your lab tech to double check your set up.

17. Allow 30 minutes for the gel to polymerize. Thaw your protein samples on ice while waiting.

18. Once the gels are set, remove them from the casting stand and assemble in the gel box. Note: Do not remove the comb yet.

19. Add ~115ml of PAGE Running buffer to the upper chamber. The buffer level should be half way between the big and small glass plate.

20. Add enough PAGE Running buffer to the lower chamber to cover the bottom half of the gels.

21. Carefully remove the comb.

Gel Set Up:1. Calculate the volumes of sample, sample buffer, and water

needed for each tube. This should be the same as in 2.3.2. Place the remaining protein samples in the freezer.3. Boil samples for 5 minutes right before you are ready to load.

After boiling, keep samples on ice while loading gel. 4. Load you samples into the gel in the following order (use gel

loading tips): Lane 1, 6: Prestained protein marker (20 L per lane) Lanes 2, 7: TransformedLanes 3, 8: Transformed plus IPTG Lanes 4, 9: Control (non- transformed)Lanes 5, 10: Control (non- transformed) plus IPTG

5. Run the gel at 50mA for 1-1 ½ hours and then set up Western blot.

Blotting ProcedureThe transfer will be accomplished using the Hoeffer Semi Dry Transfer Apparatus. This unit transfers proteins from a polyacrylamide gel to a nitrocellulose membrane by means of a low current and low voltage transfer.

1. Rinse the anode and cathode of the transfer apparatus with dH2O. Be careful not to get the leads or interlock housing wet.

36

2. Prepare the gel for transfer. Carefully cut away stacking gel with a razor blade. Measure the gel and record the dimensions.

3. Cut a hole in a Mylar mask 2 mm smaller than the gel. Center the mask on the anode of the transfer apparatus.

4. Cut six pieces of blotting paper and one piece of nitrocellulose membrane the same size as the gel. Measure and cut carefully; they must not be larger than the gel! Make sure to notch the nitrocellulose for orientation purposes!

5. Soak the blotting paper in Western blot transfer buffer.6. Rinse the nitrocellulose membrane with dH2O, then soak it in

Western blot transfer buffer for 5 minutes.7. Put one piece of the blotting paper over the opening in the

Mylar mask. Roll a test tube over the paper 3-4 times to push all air bubbles out. You will need to use moderate pressure to be effective.

8. Repeat this process adding two more blotting paper layers.9. Add the nitrocellulose paper to the stack and roll out the air

bubbles.10. Add the gel. Do not roll. Be careful setting the gel on the

stack. Try to line it up correctly the first time as some proteins may stick to the membrane on contact and moving the gel around will affect the quality of your blot.

11. Add the last three strips of blotting paper, one at a time, carefully rolling out the bubbles each time.

**NOTE: If it is too hard to place the gel exactly on top of the nitrocellulose membrane, you can put the gel down first and then the nitrocellulose, but you need to remember to FLIP THE STACK after you have finished putting the filter paper on top so that the nitrocellulose is BELOW the gel.**12. Put the top on the transfer unit. Set a flask with 1L of

water on top to add pressure.13. Connect the short safety interlock lead on the cover to the

jack on the base. Plug the leads into the power supply.14. Turn on the power (0.8 mA per cm2 of gel surface).

Transfer for 1½ hour.15. Turn off the power supply. Disconnect the leads and

safety interlock.16. Use forceps to remove the nitrocellulose membrane.

Place it face up on a large Kimwipe. Record the orientation of the lanes, and then wrap the nitrocellulose in plastic wrap and store at -20C. Blotting papers and gel can be thrown away.

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2.7 Primary Antibody Blocking (day before 2.8)

1. Cut the nitrocellulose along the edge of the middle prestained marker so that both halves of the nitrocellulose contain the visible protein marker. Wrap one half of the blot in Saran wrap, label, and place back into the freezer.

2. Submerge the other half of the blot in 25ml of blocking solution. Rock at room temperature for 1 hour.

3. Decant the blocking solution into the sink. 4. Wash the blot with 50ml 1X TBS for five minutes, rocking.

Decant into sink. Repeat for a total of 3 five minute washings. 5. Add the primary antibody (Anti-GST produced in rabbits diluted

1:5000 in 10 ml of solution containing TBS, 0.1% Tween-20, and 1% dry non-fat milk). Add enough to submerge the nitrocellulose filter.

6. Rock in the cold room overnight.

2.8 Staining and Detection of Western Blot HAZARDOUS CHEMICAL INFO:-Amido Black Stain and Destain are flammable irritants. Wear gloves when handling and dispose of in proper waste containers. -NBT/BCIP is highly toxic. WEAR GLOVES when handling and dispose of all liquid waste containing NBT/BCIP in the appropriate waste container.

Staining with Amido Black:1. Remove nitrocellulose blot from the freezer and cover with a

minimal amount of amido black.2. Rock at room temperature for 5 minutes.3. Decant stain back into its original container then wash the blot

with amido black destain until all background color is gone.4. Decant destain into the proper waste container.5. Place the blot on Kimwipe to air dry.6. Observe the stained blot. Save it to compare to the

immunostained half. Wrap the blot in plastic wrap. Copies of the stained blot should be made for each group member. Scanning and printing the blot can achieve this.

Detection of the antibody:1. Decant the primary antibody into the sink.2. Wash the blot with TBS-T for 5 minutes, rocking. Decant the

TBS-T into the sink. Repeat 2 more times. 3. Add the secondary antibody (Goat anti-rabbit IgG alkaline

phosphatase conjugated diluted 1:10000 in TBS-T).

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4. Rock the blot at room temperature for 1 hour. Save a small volume (500L) of the secondary antibody in a 1.5 ml tube to use as a control, keep on ice.

5. Decant the rest of the secondary antibody into the sink. 6. Wash the blot with 50 ml of TBS-T. Rock at room temperature

for 5 minutes.7. After 5 minutes, decant the TBS-T into the sink, add fresh TBS-T

and repeat for a total of (6) 5 minute TBS-T washings.8. Add 500 L of the NBT-BCIP to the secondary antibody you

saved. A blue color should develop within a couple minutes.9. Add 10 ml NBT-BCIP solution directly to your blot. Agitate until

color develops. (Should occur within 1 to 10 minutes).10. Once color has developed, decant the NBT-BCIP solution

into its appropriate waste container. Rinse the nitrocellulose with dH2O. Decant into the sink.

11. Air dry the nitrocellulose on a Kimwipe. Observe and record. Wrap the nitrocellulose in plastic wrap and store in your notebook. Copies of the blot should be made for each group member.

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Module 3Human Forensic DNA Analysis

Introduction This module allows you to analyze some of your own DNA for markers that are used in forensic analysis and also to simulate the analysis of criminal evidence. One marker that you will use is the TPA-25 sequence from an intron of the tissue plasminogen activator gene. This is a member of the Alu family of insertions in our genomes. You will amplify regions of chromosome 8 to determine whether the TPA-25 insert is in the intron. We can screen for this insertion sequence using polymerase chain reaction (PCR) because it has no phenotypic consequence, i.e. no health or other problems arise from its presence.

We will also amplify a region of chromosome 1 called D1S80 to look for a polymorphic sequence that has a variable number of tandem repeats (VNTR). Again, we will use PCR to amplify a region and compare results among lab members to see how we can identify each one uniquely by the size (14-40 repeats) of the PCR product from this region. The sequence we will amplify is in a non-coding region and has no phenotypic consequences.

The DNA template for your PCR will be your own, from a swab of your mouth.

The following primers are used to bracket the TPA-25 locus- The forward primer:

5’-GTAAGAGTTCCGTAACAGGACAGCT-3’

The reverse primer:

5’-CCCCACCCTAGGAGAACTTCTCTTT-3’

The following primers are used for the D1S80 VNTR-

The forward primer:

5’-GAAACTGGCCTCCAAACACTGCCCGCCG-3’

The reverse primer:

5’-GTCTTGTTGGAGATGCACGTGCCCCTTGC-3’

It is important for you to pipet accurately and to carry out these experiments carefully or your results will not be reproducible and definitive – not very good evidence for a crime scene investigation.

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3.1 Part A: To Make Template DNA:

1. Rinse your mouth with water to dislodge any debris from lunch.2. You will be assigned a number by the TA. This is your sample

identification for this lab.3. Label a 50ml tube containing 0.9% saline with your number.4. Pour all of the saline solution into your mouth and swish

vigorously for 60 seconds or more. 5. Return saline to the tube and replace cap.6. Load your tube into the IEC Centra and centrifuge at 2,800 rpm

for 10 minutes. 7. Carefully decant the supernatant into the sink; do not disturb

the pellet. Place the tube on ice.8. Resuspend the pellet in 500 L 10% Chelex solution. Note:

When dealing with Chelex make sure to use the wide-mouth pipette tips.

9. Transfer 500 L of the resuspended Chelex/cell solution to a sterile 1.5ml microfuge tube labeled with your name.

10. Incubate your sample in a boiling water bath for 10 minutes.

11. Remove your sample from the water bath and cool on ice for ~1 minute.

12. Spin your sample in the microcentrifuge for 30 seconds to pellet the Chelex beads.

13. Transfer 200 L of the supernatant to a clean, sterile 1.5 ml tube labeled with your number and place it on ice. Do not transfer any of the Chelex.

14. Waste is biohazardous! Discard appropriately.

Part B: Set Up the PCR:1. Label the cap of a 0.5 ml PCR tube with your initials.2. Use the amplification chart on the following page as a checklist

while adding reagents to the 0.5 ml PCR tube. Do not cross contaminate! Note: Keep all your tubes on ice while adding materials to them. Keep them on ice until you are ready to load into the thermocycler.

3. Add the following reagents accurately:

Reagents (uL)Tube 1 TPA-25

Tube 2 TPA-25 Tube 3 D1S80 Tube 4 D1S80

Template DNA 3 3 3 3

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10X PCR buffer 5 5 5 525 mM MgCl2 3 3 3 31.25 mM dNTPs 8 8 8 8Forward Primer 1 0 1 1Reverse Primer 1 1 1 0Taq polymerase 0.5 0.5 0.5 0.5Sterile dH2O 28.5 29.5 28.5 29.5(total volume/tube= 50 ul)

Make sure you put the correct primers in their corresponding tubes! You will be given 4 different primers: Forward & Reverse for D1S80 and Forward & Reverse for TPA-25.

4. Cap your tubes and spin them briefly to bring all the liquid to the bottom of the tube. Note: Only spin tubes briefly (5 sec.), 0.5 ml tubes are thin-walled and can crack if microfuged for too long.

5. Place tubes in the thermocyclers. Select the appropriate protocol for each set of primers.

6. After preparing the tubes for the PCR, store your remaining DNA in a labeled tube at -20C.

TPA-25 PCR Conditions 94C 5 min Initial Denature94C 1 min

30X56C 1 min72C 1 min72C 10 min Final

Elongation4C HOLD ****************

D1S80 PCR Conditions94C 5 min Initial

Denature94C 1 min

30X68C 1 min72C 1 min72C 10 min Final

Elongation

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4C HOLD ****************3.2 Preparation of 2% Agarose Gel HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Prepare a 2% agarose gel. Note: The 2% agarose solution will solidify quickly! Pour gel while still relatively hot.

2. Remove 20µL of PCR product from each tube; add to 4 µL of sample buffer. Store the remaining PCR product at 4ºC.

3. Obtain an aliquot of 100bp ladder from the Lab Tech; this will be loaded directly on the gel.

4. Once your gel is set, remove the comb and place the gel in the running box.

5. Cover the gel with 1X TAE buffer and load your DNA samples.6. Run gel at 120 volts for 45 minutes to 1 hour.7. Once the electrophoresis is complete, stain your gel for ~15

minutes in ethidium bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.

8. Destain, examine, and photograph gel.

3.3 Interpreting your results____________________________________Examine your results and consult additional literature. Draw preliminary conclusions. Your TA will ask you to describe and explain your gel.

3.4 to 3.5 Repeating PCR When dealing with forensic science, tests need to be repeated to make sure the results are the same and therefore correct. In 4.1 and 4.2, you isolated your own DNA, used PCR to amplify it, and ran it on a gel. Now, you will use the remaining isolated DNA and repeat PCR using varying amounts of water, MgCl2, dNTPs, primers, template DNA, 10X PCR buffer, and Taq. Use your past knowledge and experience of PCR to calculate the amounts and concentrations of the PCR reagents to use.

3.6 to 3.7 Murder Investigation You are a lab technician working at a forensics lab. A DNA sample has come in. The sample is evidence that has come from a murder

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scene and the murderer is unknown. The DNA sample was collected from a bottle found at the crime scene. The investigators have swabbed the bottle and placed the DNA in a sterile saline solution to preserve it until processing. Investigators have also collected DNA samples from 6 murder suspects. You must process and compare the DNA sample found at the crime scene to those collected from the murder suspects and the victim. From your results, determine who is guilty of the crime and prove why they are guilty.

In addition, other forensics labs are processing the same evidence/samples. Your results should match up with all other labs if they are correct. If your results do not match up with the other forensics labs’ results, trouble shoot and repeat until all labs have the same results. Follow the protocols that you have just used with your own DNA to prepare the DNA samples for PCR.

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Module 4Proteomics

Introduction In this module, you will compare the proteins found in wild type and mutant paramecia cilia. You will run a one dimensional polyacrylamide gel, cut out the bands of interest, and compare the proteins present in those bands using mass spectrometry.

4.0 Preparation of Cilia (Done for you)

Your Lab technician will prepare the cilia from paramecium using the following protocol:

1. Filter cell culture by slowly pouring paramecium cell culture through a funnel lined with cheese cloth sandwiched between 2 large kimwipes into a clean 2800mL flask.

2. Centrifuge cell culture to concentrate the cells. 3. Pipette cells into a beaker containing 100ml of room

temperature Dryl’s solution. Mix the solution and then pour into a clean pear-shaped flask. Centrifuge for 2 minutes at ¾ speed. Remove trichocysts (fluffy layer on top of pellet) and put in waste container. Transfer cells to clean Dryl’s buffer. Mix and centrifuge again. Do for a total of 3 washes.

4. Add cells to a flask containing 50ml Dryl’s solution, then add 50ml cold STEN buffer. Keep on ice for 10 minutes. The cells should be immobilized within 10 minutes.

5. Add 20ml Cilia Shock Buffer to cells and place on ice for 2-5 minutes. During this time the cells will deciliate – examine under inverted microscope to watch the progress of deciliation, and to make sure that the cell lysis is minimal (preferable <1%). If there is too much lysis, discard cells and start over.

6. Transfer cell solution to pear-shaped flasks and centrifuge for 2 minutes on full speed. Pour off supernatant into clean pear-shaped flask, leaving cell bodies in old flask. Spin supernatant again for 2 minutes at full speed.

7. Transfer supernatant to 30ml Cortex tubes with rubber sleeves. Spin in Beckman J2-21 Centrifuge at 14,500 rpm for 20 minutes.

8. Pour off supernatant and resuspend each pellet in 500ul membrane buffer. Consolidate pellets into one tube. Add 10ul

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Leupeptin, 10ul Pepstatin, and 20ul PMSF to tube. Spin in Beckman J2-21 centrifuge again at 14,500 rpm for 20 minutes. Resuspend pellet in 500ul membrane buffer and transfer to a 1.5ml tube. Add 1ul Leupeptin, 1ul Pepstatin, and 10ul PMSF to tube.

9. Store at -80C for future use. 10. Use ultra centrifuge to spin down cilia prior to proteomics

work.

4.1 Preparation of Resolving Gel HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. -N-Butanol is flammable. Use caution.

1. Obtain a large and small glass plate; wipe with methanol and a Kimwipe until you hear a “squeaky” noise. Handle glass plates at edges. Wear gloves!

2. Assemble the gel casting apparatus (See diagram in Appendix K). Assemble on a flat surface and then tighten screws.

3. Insert the comb and use a Sharpie to draw a line across the glass 0.7cm below the comb. Once the line is drawn, remove the comb.

4. Test to see if the apparatus is leak-proof. Squirt some water in between the glass plates and look for leaks. If leaks occur, a tighter seal must be achieved. Placing several layers of paper towels on top of the rubber seal can do this. Make sure to remove the water before pouring the gel. Use a Kimwipe to remove water droplets from between the plates of glass.

46

Before you pour your gel, have the TA or lab tech check your apparatus!

5. Prepare the resolving gel in a 15 ml tube according to the following directions. This recipe makes enough for two gels, so work in groups of 4. DO NOT ADD THE FRESHLY MADE AMMONIUM PERSULFATE UNTIL YOU ARE READY TO POUR THE GEL

Sterile dH2O 3.29 ml 4X resolving Buffer pH 8.9 2.60 ml

30% Acrylamide stock 4.00 ml TEMED 10 L

Last: Fresh 10% Ammonium persulfate 100 L

6. Gently swirl the solutions to mix WELL. 7. Using a Pasteur pipette, pour the gel by allowing the

acrylamide solution to run down along the side of the spacer. Add the acrylamide solution until it is just barely above your Sharpie line. Try to avoid making bubbles.

8. Overlay the acrylamide with N-Butanol to aid in the polymerization. Do this by gently adding the N-Butanol with a glass Pasteur pipette. You will be able to see a distinct line between the N-Butanol and the 4X Resolving solution.

9. Allow the gel to polymerize for AT LEAST 30 minutes (Any extra acrylamide mix in your tube will be a good gauge for polymerization. Make sure the cap is on).

10. Once your gel has polymerized, pour off the N-Butanol into the sink and rinse with dH2O. Add a layer of dH2O on top of your gel using a Pasteur pipette.

11. Wrap the gel/casting apparatus in a damp paper towel and then plastic wrap. Label appropriately. Store in the cold room.

4.2 Pierce Protein Assay Pierce Protein Assay (See directions in Appendix L)

Use the chart provided to develop a standard curve using BSA standards and to determine protein concentration.

1. Dilute protein sample: Make 10X and 20X dilutions for each protein sample. For example, to make a 20X dilution, add 5L of your sample to 95L of sterile dH2O. For a 10X dilution add 10L of your sample to 90L of sterile dH2O.

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2. Make dye solution: Use Solutions A and B from the Pierce Protein Assay Kit. They should be mixed 50:1…but make up only the amount you will need. Mix the dye in a 50 ml tube.

3. Add BSA and dH20 according to the directions in Appendix J.4. Add 2ml of the dye to each one of your samples and standards.5. Incubate at 37C for 30 minutes. 6. Get OD values for standards and samples: TA will assist in

the operation of the spec. 7. Place your standard into a clean cuvette.

To clean the cuvette, rinse with dH2O. Make sure to dry the outside of the cuvette with a Kimwipe. Handle the cuvette only on the frosted sides.

8. Read OD at 562nm. 9. Repeat for each standard and sample, including duplicates.

If only using 1 cuvette, make sure to rinse with dH2O between each standard.

10. Once the standards are complete, read your samples. You should blank the instrument with dH2O and dye. If using only 1 cuvette, make sure to rinse with dH2O between each sample.

11. Find the average of each standard and sample duplicates.12. Establish a standard curve using the OD values

obtained with your BSA standards: graph Concentration (x-axis) vs. OD (y-axis) on graph paper. Using this graph, calculate the protein concentrations in your three samples.

13. Use Excel to plot your data on a second chart (This is homework). Make sure to paste your Excel chart in your notebook properly labeled.

4. 3 SDS-PAGE HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the APS) in the appropriate waste container.

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-Coomassie Blue Stain and Coomassie Blue Destain are highly flammable and irritating to the skin. WEAR GLOVES when handling and dispose of in appropriate waste container.

22. Remove the resolving gel from the cold room and pour a 4% stacking gel.

23. Mix the following components in a 15 ml tube (makes enough for 2 gels).

24. Sterile dH2O 6.10 ml 4X stacking buffer pH 6.8 2.50 ml

30% Acrylamide stock 1.30 ml TEMED 10 L

Last: Fresh 10% Ammonium persulfate 50 L

25. Before adding the ammonium persulfate, pour the dH2O off the resolving gel and dry with a Kimwipe.

26. Add the ammonium persulfate to your tube. Mix gently.27. Pour the stacking gel as you did the resolving gel all the

way to the top of the small glass plate. If it overflows when inserting the comb this is okay. Clean the comb thoroughly with methanol before inserting.

28. Being careful to avoid making air bubbles, insert the clean comb until there is no air between the wells. This is VERY IMPORTANT; the stacking gel will not polymerize if the comb is not clean, or if there is air between the wells. Ask your lab tech to double check your set up.

29. Allow 30 minutes for the gel to polymerize. Thaw your protein samples on ice while waiting.

30. Once the gels are set, remove them from the casting stand and assemble in the gel box. Note: Do not remove the comb yet.

31. Add ~115ml of PAGE Running buffer to the upper chamber. The buffer level should be half way between the big and small glass plate.

32. Add enough PAGE Running buffer to the lower chamber to cover the bottom half of the gels.

33. Carefully remove the comb.

Gel Set Up11. Calculate the volumes of sample, sample buffer, and water

needed for each tube (make 60µl total so that even if some evaporates during boiling, there will still be 40µl left):

-Each tube needs 60µg of protein total. Using the concentrations you calculated off of your standard curve, calculate how many microliters equals 60µg.

49

-Each tube needs 1X SDS sample buffer. You are given 6X SDS sample buffer. Calculate how much 6X SDS sample buffer is needed so that the final concentration is 1X.-Each tube needs a total volume of 60µl. Figure out how much water should be added to each so that the total volume is 60µl.

12. Add the calculated volumes of water, protein, and 6X sample buffer to a 1.5ml tube. Place the remaining protein samples in the freezer.

13. Boil samples for 5 minutes right before you are ready to load. After boiling, keep samples on ice while loading gel.

14. Load you samples into the gel in the following order (use gel loading tips): Lane 1: 6X SDS sample buffer/Blank Lanes 2, 5: Prestained protein marker (20 L per lane) Lanes 3, 6: Treatment 1 Lanes 4, 7: Treatment 2

15. Run the gel at 50mA for 1-1 ½ hours.16. Remove gel carefully from the gel apparatus. Use a razor

blade to cut the stacking gel portion away. Dispose of the stacking gel in the appropriate waste container.

17. Put the gel into a plastic container and cover with Coomassie stain. Microwave on low for 1 minute. Discard stain in appropriate waste container.

18. Transfer gel to destain. Wash and discard destain in correct waste container.

19. Rinse gel with destain again and decant destain into correct waste container.

20. Submerge the gel in more destain. Rock gently overnight. 21. The Lab tech will save these gels in destain until the next

lab.

4.4 Cutting out gel bands and trypsinizing proteins in preparation for Mass SpectrometryHAZARDOUS CHEMICAL INFO:-Acetonitrile is poisonous. It also tends to leak from pipet tips when being measured. Always wear eye protection and gloves when handling it or transferring it. Always dispose of acetonitrile in its special waste receptacle. If you get acetonitrile on your gloves, change your gloves.

1. Clean a glass plate with soap and water and rinse thoroughly with ddwater. Rinse again with 70% ethanol. Let the plate dry.

2. Carefully remove the gel from its container and place on the glass plate.

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3. Label a 1.7 ml microcentrifuge tube with appropriate identification for each protein band to be analyzed. Select the appropriate sized scalpel for the band to be removed and clean with 70% ethanol before using and between cutting each band. When choosing a scalpel select one that will minimize the amount of gel you take with it.

4. Select bands of interest based on visible differences in intensity of Coomassie staining. Cut out the selected gel bands and transfer them to the labeled tubes carefully.

5. Add 900 ul of HPLC-grade water to each tube. Incubate at room temperature for five minutes.

6. Centrifuge at high speed for 30 seconds. Using your pipettor set to 1000 ul, carefully remove the water and discard it in the waste container provided. Use a new pipet tip for each sample. Be careful not to lose the gel in this process.

Note: The following steps use acetonitrile, which is poisonous. It also tends to leak from pipet tips when being measured. Always wear eye protection and gloves when handling it or transferring it. Always dispose of acetonitrile in its special waste receptacle. If you get acetonitrile on your gloves, change your gloves.

7. Add 750 ul destain solution (50mM ammonium bicarbonate, 50% acetonitrile) to each tube. Close the tube cap and mix gently by inversion. Incubate the tubes at 37 degrees C for 20 minutes.

8. At the end of 20 minutes inspect the gel. If it is still very blue, repeat the destain process with more destain reagent. To do this, briefly centrifuge the tube, carefully pipet off the liquid, add 750 ul new destain reagent and incubate for an additional 15 minutes. Make sure you put the discarded destain solution in the provided waste receptacle. Repeat one more time if needed. Change gloves if you get acetonitrile on them and wash skin with water if it contacts the acetonitrile.

9. When the gel sample is clear and no longer contains blue color, centrifuge the tube at high speed for 30 seconds. Carefully remove all the destain solution and discard it in the waste receptacle.

10. Add 100 ul of 100% acetonitrile to each tube. (The gel pieces should be entirely immersed, if not add more). The gel pieces will turn white as they dehydrate. Incubate the tubes for 2 minutes at room temperature.

11. Centrifuge for 30 seconds and carefully pipet off all the acetonitrile. Discard in the provided waste receptacle. Use a 200 ul pipettor to carefully remove the residual liquid from the tube.

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12. Keep the tube lid open, lay the tube on its side and let the gel dry completely for 5 minutes at room temperature. Close the lid before proceeding to the next step.

13. Place the tubes on ice for 5 minutes. Add 25 ul ice cold trypsin/50mM ammonium bicarbonate solution to each tube. Incubate on ice for five minutes. Add 25ul cold 50mM ammonium bicarbonate solution to each tube. Make sure the gel is completely immersed in the solution. Add more ammonium bicarbonate solution if necessary.

14. Incubate the tubes on ice for an additional 30 minutes. At this point tubes may be processed following steps 14 and

15 or can be transferred directly to UVM for further processing. They do not need to be kept on ice, but packaged so the contents will not spill.

15. Transfer the tubes to a 37o C incubator. Incubate the tubes overnight (8-16 hrs).

16. Store the tubes at 4o C until ready for further analysis.

4.5 Mass Spec

Bring your prepared samples to the UVM Proteomics Core Facility at MLS 311. Tour the facility and meet with Dr. Ballif to discuss how mass spec works.

4.6 Analyze results

Your TA and professor(s) will help you to analyze your results from mass spec, and determine which proteins are differentially expressed.

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Module 5RNA Interference

Introduction In this module, a specific target gene product of Paramecium tetraurelia is depleted using an RNAi feeding method. Paramecia are fed with an RNAase III-deficient E. coli strain, HT115 bacteria transformed with a gene specific RNAi construct. The RNAi construct has T7 RNA polymerase promoters on either side of the cloned gene which expresses double stranded RNA upon IPTG induction. After 48 hours of feeding at 280C, the level of target endogenous mRNA is determined by semi-quantitative Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR).

The lab tech has transformed E.coli HT115 cells. The “test” cells contain a plasmid with the pawn A gene, while the “control” cells contain a plasmid without the pawn A gene. Both the test and control cells are resistant to ampicillin and tetracycline.

5.0 (Done for you) ON Bacterial Culture The lab tech will start ON cultures from freshly transformed HT115 cells. The cells will grow with shaking at 37C overnight.

5.1 Induce Bacteria and Feed to Paramecium Part A: Inducing bacteria

1. Two hours before lab, the lab tech will add 1 ml of the test and control cells previously grown ON to 50 ml LB amp medium.

2. The cells will shake at 37C for two hours.3. After two hours of shaking measure the OD value of the culture

at 595nm using the spectrophotometer. When the OD value reaches between 0.3 and 0.4, proceed to the next step.

4. Add 125ul 50mM IPTG to each 50ml bacterial culture. Shake at 37 C for 3 hours.

Part B: Purging Paramecium Cells(Your TA will demonstrate procedure in MLS 226)

1. Obtain a 100ml flask of paramecium cells grown in bacterialized wheat culture from the 28C incubator. (These cells were added to the bacterialized wheat culture 4 days earlier and are now feeling starved.)

2. Pour cells through a small folded Kimwipe nestled inside a small funnel atop a 100ml pear-shaped flask.

3. Balance the flask with a flask filled with water and centrifuge at ¾ speed for 2 minutes in the IEC HN-SII centrifuge.

4. Obtain a 15ml plastic centrifuge tube containing Dryl’s solution.

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5. After centrifugation, your TA will demonstrate how to transfer the cells from the bottom of the pear-shaped to the Dryl’s solution using a glass Pasteur pipette.

6. Keep the cells in the Dryl’s solution until you are ready to add them to the flasks containing your test and control bacterial cultures.

Part C: Preparing the bacterial cultures for the paramecium1. After three hours of shaking, transfer the test and control

bacterial cultures to large, sterile centrifuge bottles and harvest the bacteria by centrifuging at 5000 rpm, 4ºC for 10 minutes in the Beckman J2-21.

2. Decant the supernatant broth into the waste jar.3. Resuspend each bacterial pellet in 100ml wheat culture

containing 100ul Amp, 100ul Stigmasterol, and 250ul 50mM IPTG. Transfer resuspended pellet to flask.

4. Transfer approximately 1ml of paramecium cells in Dryl’s solution to a depression slide under a dissecting microscope. Using a Pasture pipette count and transfer 350 paramecium cells to each flask of culture.

5. Place flasks in 28C incubator.

5.2 (Done for you) Induce paramecium culture After 24 hours of incubation, your TA will add 100ul Stigmasterol and 250ul 50mM IPTG to each of the cultures and continue to incubate at 28C.

5.3 Harvest cells and Isolate RNA HAZARDOUS CHEMICAL INFO:2-Mercaptoethanol is toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the 2-Mercaptoethanol) in the appropriate waste container. Do not inhale fumes. Part A: Checking Paramecium PhenotypesThe TA will describe the expected phenotypes of the Test and Control cells. View the cells in a depression slide under a dissecting microscope to confirm the expected phenotypes of both treatments.

1. Transfer approximately 1ml of cells from the culture flask to a depression slide containing resting solution. Do this for each of the two treatments. Rinse pipette in dH2O between treatments. Let the cells sit in the resting solution for 9-10 minutes.

2. Transfer a few cells at a time to a depression slide containing 5mM BaCl2. Observe the phenotypes of the control and test treatments. If the paramecium phenotypes are the expected

54

phenotypes for each treatment, continue on to filter and wash the cells.

Part B: Filtering and Washing the Paramecium (Your TA will demonstrate procedure in MLS 226)

1. Filter cells through a folded square of small Kimwipe into a 100ml pear-shaped flask

2. Balance flasks and centrifuge at ¾ speed for 1 minute in the IEC HN-SII centrifuge.

3. Using a glass Pasteur pipettes remove the cells collected at the bottom of the flask. (Your TA will demonstrate how to use circular motion to aid in removing concentrated amount of cells). Dump the supernatant into the waste container provided and then place the pipette containing the cells back into the empty pear-shaped flask.

4. Add approximately 100ml Dryl’s solution to the empty pear-shaped flask and pipette in the cells to mix.

5. Balance and centrifuge the flasks at ¾ speed for 1 minute in the IEC HN-SII centrifuge.

6. Pipette out cells using the demonstrated technique and transfer to a 1.5ml microcentrifuge tube. Continue to next section and start RNA Prep within a few minutes of collecting cells.

Part C: RNA Prep (To be completed in MLS 319 by students) 1. Disinfect lab bench, pipettes, Sharpie, and gloves of all RNases.

Spray surfaces with “RNase Away” and then wipe with paper towel. It is crucial that the following protocol be performed in an RNase-free environment. Your TA will explain the importance of RNase-free conditions.

2. Centrifuge cells for 10 seconds and remove the supernatant using a pipette. Transfer approximately 100ul of the tightly packed cells to a 0.5ml centrifuge tube.

3. Add 350L of the lysis buffer (RA1) to the 0.5ml tube of cells4. Next, add 3.5 L of 2-Mercaptoethanol. Use 2-

Mercaptoethanol in the hood and place all pipette tips that come into contact with 2-Mercaptoethanol into designated waste container!

5. Use a 1ml syringe to mix the solution6. Place a pink filter column inside a collecting tube and pipette

the cell solution onto the center of the filter. Label both the collecting tube and the filter.

7. Centrifuge for 1 minute at 12,000 rpm8. Remove filter and discard9. Add 350L of 70% ethanol to the sample in the collecting tube.

Pipette up and down to mix10. Set up a blue spin column with collecting tube and label

both.

55

11. Transfer the sample to the blue spin column and centrifuge for 1 minute at 8,000 rpm

12. Place column into a new collecting tube and label. Discard supernatant and used collecting tube.

13. Add 350L Membrane Desalting Buffer (MDB) and centrifuge at 12,000 rpm for 1 minute

14. Add 90L of DNase Reaction Buffer and 10L of DNase I stock solution to a fresh 0.5ml microcentrifuge tube

15. Take 95L of the DNase Reaction Mixture and add it to the center of the blue spin column. Let sit at room temperature for 25 minutes

16. Add 200L RA2 Buffer to the spin column and centrifuge for 1 minute at 8,000 rpm. (If the tip of the column outlet comes into contact with the flow-through for any reason, discard the flow-through and centrifuge again at 12,000 rpm for 1 minute.)

17. Place spin column into a new collecting tube18. Add 600L of RA3 Buffer to spin column and centrifuge for

1 minute at 8,000rpm19. Discard the flow-through and place the column back into

the collecting tube. 20. Add 250L RA3 Buffer to the spin column and centrifuge

for 2 minutes at 12,000 rpm.21. Place the spin column into the supplied RNase-free 1.5ml

microcentrifuge tube and label. 22. Elute the RNA by adding 20L of RNase-free water

directly into the center of the spin column. Centrifuge for 1 minute at 12,000 rpm

23. Take the liquid collected in the collecting tube and transfer to the top of column to elute again. Wait 1 minute and then centrifuge at 12,000 rpm for 1 minute.

24. Discard spin column and place tube in the -80C freezer.

5.4 Making cDNA Your TA will determine the total RNA yield for each of your samples using the Nanodrop. The concentration will be reported in ng/L.

1. Calculate the volume of RNA needed to equal 5ug per tube using the RNA concentration provided by the TA.

2. Disinfect lab bench,3. pipettes, Sharpie, and gloves of all RNases. Spray surfaces

with “RNase Away” and then wipe with paper towel. It is crucial that the following protocol be performed in an RNase-free environment.

4. Set up the RT-PCR using 0.5ml tubes and the following table:

56

Reagents (uL)Tube 1 (Test)

Tube 2 (+ Control)

Tube 3 (- Control)

RNA * 5ugWater *50uM Oligo dT 1 1 110 mM dNTPs 1 1 1Total volume 13 13 13

*Your TA will inform you on how much RNA to add to each tube based on the results from the Nanodrop. Calculate the volume of water that you need based on the amount of RNA added. Add this chart to your lab notebooks with calculated volumes.

5. Incubate @65C for 5 minutes then put on ice and add:

Reagents (uL)Tube 1 (Test)

Tube 2 (+ Control)

Tube 3 (- Control)

5X First-Strand Buffer 4 4 40.1M DTT 1 1 1RNase Out 1 1 1SuperScript III 1 1 0Water 0 0 1Total Volume 20 20 20

6. Place tubes in thermocycler and set to the following conditions:

cDNA synthesis Conditions 25C 5 min50C 30 min55C 30 min70C 15 min

7. Remove tubes from thermocycler and place at -20C.

5.5 Template Control and Endogenous RT-PCR Part A: Serial Dilutions

1. Make serial dilutions of both the Test and +Control cDNA according to the following chart:

Serial Dilution cDNA H2O1:10 2 ul of cDNA 18 ul

57

1:50 2 ul of previous dilution

8 ul

1:100 4 ul of previous dilution

4 ul

1:500 2 ul of previous dilution

8 ul

2. Keep tubes on ice while preparing PCR tubes

Part B: Template Control RT-PCRCalmodulin is a protein highly expressed in paramecium. We will detect the mRNA levels of the calmodulin gene in both the Test and +Control to determine if there are equal concentrations of mRNA in both the Test and the +Control.

Calmodulin primers: Forward: 5’ CTG AAG CTG AAC TTC AAG 3’Reverse: 5’ TCA TTT AGA AAC CAT CAT TCT 3’Product length: 330-350 bp

1. Set up and label 9 0.5ml tubes according to the table below.

Calmodulin RT-PCRTube

Treatment ul of cDNA

ul of Master Mix

1 1X Test 1 492 1:10 Test 1 493 1:100 Test 1 494 1:500 Test 1 495 1X +Control 1 496 1:10

+Control1 49

7 1:100 +Control

1 49

8 1:500 +Control

1 49

9 1X -Control 1 49

2. Each tube will contain 1ul of the respective cDNA dilution and 49ul of master mix. The master mix contains the reagents necessary for the PCR. Make the master mix in a 1.5ml tube by adding:

Sterile dH2O 305ul10X PCR buffer 50ul25mM MgCl2 30ul

58

1.25 mM dNTPs 80ulCalmodulin F primer 10ulCalmodulin R primer 10ulTaq Polymerace 5ul

3. Mix the solution well by pipetteing up and down.4. Add 49ul of master mix to each labeled tube. 5. Add 1ul of respective cDNA dilution to each tube. Mix well.6. Place tubes in PCR machine making sure the caps are completely

closed and the contents of the tube are at the bottom of each tube and not stuck up on the sides or in the cap.

7. Run PCR with the following conditions:

Calmodulin RT-PCR Conditions 95C 5 min Initial Denature95C 1 min

24X42C 1 min72C 40 sec72C 10 min Final

Elongation4C HOLD ****************

Part C: Endogenous gene RT-PCR

The endogenous PCR will allow us to check the mRNA levels and see if the gene was down regulated due to RNA interference.

Endogenous gene Primers:Forward: 5’ TAC TTG CGA ACA ATA TTA TCA 3’Reverse: 5’ ATG AAA AAC AAC AGA AAA GTA TAG 3’Product length: 327 bp

1. Set up and label 9 0.5ml tubes according to the table below.

Endogenous RT-PCRTube

Treatment ul of cDNA

ul of Master Mix

1 1X Test 1 492 1:10 Test 1 493 1:50 Test 1 494 1:100 Test 1 495 1X +Control 1 496 1:10

+Control1 49

59

7 1:50 +Control

1 49

8 1:100 +Control

1 49

9 1X -Control 1 49

2. Each tube will contain 1ul of the respective cDNA dilution and 49ul of master mix. The master mix contains the reagents necessary for the PCR. Make the master mix in a 1.5ml tube by adding:

Sterile dH2O 305ul10X PCR buffer 50ul25mM MgCl2 30ul1.25 mM dNTPs 80ulEndogenous F primer 10ulEndogenous R primer 10ulTaq Polymerace 5ul

3. Mix the solution well by pipetteing up and down.4. Add 49ul of master mix to each labeled tube. 5. Add 1ul of respective cDNA dilution to each tube. Mix well.6. Place tubes in PCR machine making sure the caps are

completely closed and the contents of the tube are at the bottom of each tube and not stuck up on the sides or in the cap.

7. Run PCR with the following conditions:

Endogenous RT-PCR Conditions 94C 2 min Initial Denature94C 1 min

30X55C 2 min72C 1 min72C 15 min Final

Elongation4C HOLD ****************

8. When the PCR is complete your TA will remove the PCR product tubes from the thermocycler and place at 4C.

5.6 Template Control Gel HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

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1. Make 300ml 1X TAE.2. Prepare a 2% agarose gel with a 12-well comb. Note: The 2%

agarose solution will solidify quickly! Pour gel while still relatively hot.

3. Remove 10µL of PCR product from each tube; add to 2 µL of sample buffer. Store the remaining PCR product at 4ºC.

4. Obtain an aliquot of 100bp ladder from the Lab Tech; this will be loaded directly on the gel.

5. Once your gel is set, remove the comb and place the gel in the running box.

6. Cover the gel with 1X TAE buffer and load your samples.7. Run gel at 120 volts for 45 minutes to 1 hour.8. Once the electrophoresis is complete, stain your gel for ~15

minutes in ethidium bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.

9. Destain, examine, and photograph your gel.

5.7 Endogenous Gel HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 300ml 1X TAE.2. Prepare a 2% agarose gel with a 12-well comb. Note: The 2%

agarose solution will solidify quickly! Pour gel while still relatively hot.

3. Remove 10µL of PCR product from each tube; add to 2 µL of sample buffer. Store the remaining PCR product at 4ºC.

4. Obtain an aliquot of 100bp ladder from the Lab Tech; this will be loaded directly on the gel.

5. Once your gel is set, remove the comb and place the gel in the running box.

6. Cover the gel with 1X TAE buffer and load your samples.7. Run gel at 120 volts for 45 minutes to 1 hour.8. Once the electrophoresis is complete, stain your gel for ~15

minutes in ethidium bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.

9. Destain, examine, and photograph your gel.

61

Appendix A: Solutions Guide

GENERAL SOLUTIONSAmpicillin Stock

100 mg/ml stock solution: for example, 0.5g ampicillin sodium salt into 5 ml dH20. Filter sterilize, and store at -20°C.

LB amp100 µg/ml final concentration: 1:1,000 dilution of ampicillin stock into LB broth.

When making LB amp plates, add 1 ml ampicillian stock (100mg/ml) into 1L LB Agar broth. *NOTE: Ampicillin is heat-sensitive, so LB agar broth must be cooled to 60°C after coming out of the autoclave, before the ampicillin is added. Setting the water bath to 60°C and letting the LB agar broth cool in there for an hour is a good way to ensure the LB agar doesn’t solidify.

When making LB amp broth, add 1ul of ampicillian stock (100mg/ml) for each 1ml of LB broth.

TE bufferNeed (final conc.): 10mM Tris-Cl (pH 7.5), 1mM EDTA (pH 8.0)Make from liquid stocks of Tris-Cl and EDTA5ml 2M Tris-Cl (pH 7.5)2ml 0.5M EDTA (pH 8)993ml dH20

2M Tris-Cl (pH 8.0)177.6g Tris-Cl10.6g Tris-baseIn ~950mL sterile dH20**Ph 8.0**Bring up to 1L with sterile dH20

0.5M EDTA (pH 8)93.05g EDTA disodium salt (FW= 372.2)In ~400ml sterile dH20***bring pH to 8.0***Bring up to 500mL with sterile dH20

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50X TAE stock (pH 8.5)242g Tris Base (FW= 121.14)In ~750ml sterile dH20Carefully add 57.1mL Glacial Acetic Acid100mL 0.5M EDTA (pH 8.0)Bring up to 1L with sterile dH20pH 8.5, but no adjustment needed*Dilute 50X TAE stock 1:10 for a 5X stock*

6X DNA sample bufferNeed: 0.25% (w/v) Bromophenol Blue

40% (w/v) Sucrose Make: 0.25 g Bromophenol Blue

40 g Sucrose100 ml dH20

λ Hind III markerWant a final concentration of 100 ng/µL from Invitrogen stock.Make: 96 µL λ Hind III marker (Invitrogen stock)

320 µL TE68 µL 6X loading buffer

20X SSC (pH 7)175.3 g NaCl88.25 g Na3 Citrate•2H201 L dH20**pH 7.0**

4X TBS (pH 7.4)60.55 g Tris base4.0 g SDS1000 ml dH20

TBS-T1X TBS, 0.1% Tween-20

2 ml Tween-20 (use large orifice tips to pick up Tween)1998 ml 1X TBS

Phosphate Buffer (PBS)4.0g NaCl0.1g KCl0.72g Na2HPO4

0.12g KH2PO4

In ~400ml sterile dH20

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**pH 7.4**Bring to 500ml with sterile dH20

MODULE 1

1.1: Isolation of plasmid DNA

Solution I0.50 g D-Glucose0.625 ml 2M Tris-Cl (pH 8)1 ml 0.5M EDTAAdd dH20 to make total volume 50ml**add 5 mg/ml lysozyme just before use**

Solution II2 ml 1M NaOH1 ml 10% SDS7 ml dH20**Prep fresh**

5M Potassium Acetate 29.5 ml glacial Acetic Acid 100 ml dH20 Add KOH pellets until pH=4.8Store in refrigerator

Heat-treated RNase A (100mg/ml)Dissolve 100 mg (0.1g) of pancreatic RNase A in 1 ml 10mM Tris-Cl/15mM NaCl. Store at -20 ºC. Before use, heat RNase A in 100°C heat block for 15 minutes and allow tubes to cool slowly to RT.

Salt Saturated PhenolTris buffered Phenol pH 6.6/7.98-Hydroxyquionoline added until dark yellow/orange color

Chloroform:Isoamyl Alcohol (24:1)480 ml Chloroform20 ml Isoamyl Alcohol

TE bufferSee general solutions section

64

1.4: Transformation

50 mM CaCl20.73 g CaCl2

100 ml dH20** Autoclave to sterilize or make fresh**

Transformation buffer1ml 100 mM CaCl2

1 ml 100 mM Tris1 ml 100 mM NaCl7 ml dH20

1.6: Secondary selection of transformed bacteria

0.145 M Sterile Saline (pH ~7)4.25 g NaCl500 ml dH20** Autoclave to sterilze**

1.8: Cracking gel

Cracking Buffer (pH 6.8) 0.788 g Tris-Cl1.0 g SDS0.058 g Na2EDTA•2H2013.6 g Sucrose0.1 g Bromophenol Blue100 ml dH20** pH 6.8**

1.9: Biotin labeling of DNA

3 M Sodium Acetate (pH 4.8)24.6 g Sodium Acetate100 ml dH20** pH 4.8**

1.11: Southern blot

Cracking BufferSee 1.8 above

65

0.5M NaOH/0.8M NaCl20 g NaOH46.752 g NaCl1 L dH20

0.5M Tris/1.5M NaCl (pH 7)250 ml 2 M Tris-base solution87.6 g NaCl750 ml dH20** pH 7**

10X SSCSee general solutions for 20X SSC

1.13: Hybridization of Southern Blot

Prehybridization Solution (per group, prep fresh)5 ml Formamide2.5 ml 20X SSC0.5 ml 100X Denhardt’s solution (doesn’t keep more than 24

hours!)0.25 ml 1M Phosphate Buffer200 µL Herring sperm DNA (2mg/ml, made fresh), freshly

denatured

Hybridization Solution (per group, prep fresh)4.5 ml Formamide 2.5 ml 20X SSC0.1 ml 100X Denhardt’s solution (doesn’t keep more than 24

hours!)0.4 ml 1M Phosphate Buffer1.5 ml dH2O200 µL Herring sperm DNA (2mg/ml), freshly denaturedBiotin-labeled probe DNA

100X Denhardt’s Solution0.2 g Ficoll 0.2 g Polyvinylpyrrolidone0.2 g Bovine Serum Albumin (BSA)10ml sterile dH20**Doesn’t keep more than 24 hours**

1.14: Detection of DNA

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2X SSC/0.1% (w/v) SDS (per group)50 ml 20X SSC450 ml dH200.5 g SDS

0.2X SSC/0.1% (w/v) SDS (per group)5 ml 20X SSC495 ml dH200.5 g SDS

0.16X SSC/0.1% (w/v) SDS (per group)4 ml 20X SSC496 ml dH200.5 g SDS

1.15: Development of Blot

Buffer 1 : Final Concentration: 0.1 M Tris-Cl 0.15 M NaCl

8.7 g NaCl15.764 g Tris-Cl1 L dH20

Buffer 2: 3% (w/v) BSA in Buffer 13g BSA per 100 ml Buffer 1**Doesn’t keep more than 24 hours, prep fresh**

SA-AP: **Needs to be made immediately before use**1 µL SA-AP per 1 ml Buffer 2 (approx. 10 ml needed per group)

Buffer 3Final concentration: 0.1M Tris-Cl

0.1M NaCl 50 mM MgCl2

15.764 g Tris-Cl5.844 g NaCl10.15 g MgCl2

1 L dH2O

1.17: PCR20 µM Primer Dilutions (from 500 µM stock primers)

5 µL 500µM stock

67

120µL dH20**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in smaller batches**

10 mM dNTP mix (from 100 mM individual dNTP stocks)500µL dCTP500µL dTTP500µL dATP500µL dGTP3 ml dH20 *1 µL of 10mM dNTP mix per 50µL reaction*

1.25mM dNTP mix262.5µL dH2037.5µL 10mM dNTP mix *8 µL of 1.25mM dNTP mix per 50 µL reaction*

MODULE 2

2.2: SDS-PAGE prep and Pierce Protein Assay100mM IPTG

0.02383g IPTG1 ml dH20

4X Resolving Buffer (pH 8.9)18.17g Tris base10 g SDS (measure in hood)100 ml dH20**pH 8.9**

Lysis Buffer5 ml 1M Tris (pH 8.0)3 ml 5M NaCl1 ml Triton 100X91 ml dH20**add 0.0057 g DTT/10 ml Lysis Buffer fresh immediately before

use**

2.3: SDS-PAGE4X Stacking Buffer (pH 6.8)

6.055 g Tris base0.4 g SDS

68

100 ml dH20**pH to 6.8**

PAGE running buffer10.92 g Tris base51.36 g Glycine3.6 g SDS1800 ml dH20

6X SDS sample buffer15 ml Glyercol6.25 ml 2M Tris-Cl0.186Na2•EDTA2 g SDS0.1 g Bromophenol Blue78.75 ml dH20** 1% (v/v) β-Mercaptoethanol needs to be added fresh before

use**

Coomassie Blue Stain200 ml Methanol50 ml Glacial Acetic Acid1 g Coomassie Blue250 ml dH20

Coomassie Blue Destain200 ml Methanol 75 ml Glacial acetic Acid725 ml dH20

2.6: SDS-PAGE and Western Blot

Western Blot Transfer Buffer (pH 8.3)2.93 g Glycine5.81 g Tris base200 ml Methanol800 ml dH20**pH 8.3**

2.7: Blocking with primary antibody

Blocking Buffer50 g dry milk

69

6.35 g Tris-Cl1.18 g Tris base8.77 g NaCl1L dH20

TBS-TSee general solutions section.

Primary Antibody Dilution (1:5,000)0.1% Tween-20, 1% (w/v) dry milk

50 ml 1X TBS50 µL Tween-200.5 g dry milk10 µL Anti-GST antibody

2.8: Staining with Amido Black and detection using secondary antibody

Amido Black Stain112.5 ml Methanol5.0 ml Acetic Acid0.25 g Amido Black132.5 ml dH20

Amido Black Destain112.5 ml Methanol5.0 ml Acetic Acid132.5 ml dH20

Secondary Antibody Dilution (1:10,000)4 µL Secondary Antibody40 ml TBS-T

MODULE 3

3.1: Making Template DNA and Performing PCR Reaction0.9% Sterile Saline Solution

9 g NaCl1000 ml dH20Autoclave to sterilize

10% Chelex Extraction Solution

70

10 g Sigma Chelex 100 Resin100 ml dH20**prep fresh**

20 µM Primer Dilutions (from 500 µM stock primers)5µL 500µM stock120µL dH20**Primers should be stored long term at 500 µM conc. and

diluted to 20 µM in smaller batches**

10 mM dNTP mix (from 100 mM individual dNTP stocks)500µL dCTP500µL dTTP500µL dATP500µL dGTP3 ml dH20

1.25mM dNTP mix262.5µL dH2037.5µL 10mM dNTP mix

MODULE 4

4.0 Preparation of CiliaDryl’s solution

1mM Na2HPO4

1mM Na2H2PO4

1.5mM CaCl2

2mM Sodium CitratepH 6.8

STEN Buffer17.12g Sucrose0.316g TrisCl0.068g Na2EDTA0.036g NaCl100ml dH2O*pH to 7.5 and fill**store at 4C

Cilia Shock Buffer 180mM KCl 60mM CaCl2

Membrane Buffer0.331g Tris-Cl

71

0.048g Tris base0.186g KCl0.051g MgCl2•6H2O0.019g EGTA50ml dH2O*pH to 7.4**add 1% Triton X-100 before use

LeupeptinDissolve in dH2O 1mg/mlStore at -20C in 1ml aliquots*Stable for 6 months

Pepstatin A Dissolve in methanol 1mg/ml(5ml methanol into 5mg bottle and shake)Store at -20C in 1ml aliquots*Stable for 1 month

100mM PMSF stock (Phenylmethylsulfonyl fluoride)0.871g PMSF50ml 100% ethanolAdd ~1.75g of molecular sieves to each bottle to absorb any

water*inactive in 30 minutes @ 4C in aqueous solution

4.1 Preparation of Resolving Gel

4X Resolving Buffer (pH 8.9)18.17g Tris base10 g SDS (measure in hood)100 ml dH20**pH 8.9**

4X Stacking Buffer (pH 6.8) 6.055 g Tris base0.4 g SDS100 ml dH20**pH to 6.8**

PAGE running buffer10.92 g Tris base51.36 g Glycine3.6 g SDS1800 ml dH20

72

6X SDS sample buffer15 ml Glyercol6.25 ml 2M Tris-Cl0.186Na2•EDTA2 g SDS0.1 g Bromophenol Blue78.75 ml dH20** 1% (v/v) β-Mercaptoethanol needs to be added fresh before

use**

Coomassie Blue Stain200 ml Methanol50 ml Glacial Acetic Acid1 g Coomassie Blue250 ml dH20

Coomassie Blue Destain200 ml Methanol 75 ml Glacial acetic Acid725 ml dH20

4.4 Band Cutting and Trypsinization of Gel PlugsTrypsin in 50mM ammonium bicarbonate

make up same day and keep on ice

MODULE 5

5.1 Induce Bacteria and Feed to Paramecium

50mM IPTG750ul dH2037.5mg IPTG

Wheat cultureWheat grass tea buffered with:3.75mM Na2HPO4+7H2O3mg/L Stigmasterol

Dryl’s solution1mM Na2HPO4

1mM Na2H2PO4

1.5mM CaCl2

2mM Sodium CitratepH 6.8

73

Stigmasterol5mg/ml dissolved in 100% ethanol

5.3 Harvest cells and Isolate RNA

Resting Solution (4.11.1) 1mM Citric acid1mM Calcium hydroxide1mM Tris base5mM KCl pH 7.04

5mM BaCl21mM Citric acid1mM Calcium hydroxide1mM Tris basepH 7.09

5.4 Making cDNA

50uM Oligo dT20uM Primer 5’ CGGCTCGAGTTTTTTTTTTTTTTTTTTTT 3’

10 mM dNTP mix (from 100 mM individual dNTP stocks)500µL dCTP500µL dTTP500µL dATP500µL dGTP3 ml dH20

5.5 Template Control and Endogenous RT-PCR20 µM Primer Dilutions (from 500 µM stock primers)

5µL 500µM stock120µL dH20**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in smaller batches**

1.25mM dNTP mix262.5µL dH2037.5µL 10mM dNTP mix

74

75

Appendix B: Sterile TechniqueIt is very important in microbiology and genetics to work with pure cultures. Unfortunately, this is difficult. The world around us is covered with microorganisms. Microorganisms are even carried on dust particles in the air. In order to protect sterile broth, plates, slants and pure cultures from the microbes all around us, we must practice sterile (aseptic) technique. This simple means that sterile surfaces or sterile media must be protected from contamination by microbes in the air or residing on non-sterile surfaces. A simple example of the problem is that a sterile petri plate can become contaminated with bacteria when the lid is removed. In sterile technique, only sterile surfaces touch other sterile surfaces and exposure to the air is kept to a minimum.

In the classroom, you often need to practice sterile technique when you inoculate a pure culture of a microorganism into fresh medium. Sometimes this is a transfer to a tube of liquid broth and at other times, it is a transfer to a petri plate-containing agar. While there are other circumstances that require sterile technique, these are the most common and they will be described in more detail on the pages that follow.

Appendix C: Spread Plate Technique

1. Dispense the appropriate volume of sample into the center of a sterile agar plate.

2. Dip the glass spreader (aka “hockey stick”) in alcohol. 3. Pass the spreader through the flame of a Bunsen burner to burn

off the alcohol. (This sterilizes the spreader).

***IMPORTANT***Keep the dish of alcohol behind the Bunsen burner.

Keep the alcohol dish covered when you are not using it.Keep your hand above the spreader at all times or flaming

alcohol may roll toward your hand.If the dish of alcohol catches on fire, cover the dish with the

glass lid and it will go out.

4. Cool the spreader by touching it to the agar where there is no sample.

5. Spread your sample over the entire surface of the agar.6. Sterilize the spreader before putting it back on the bench.

76

Appendix D: Use of a Rainin Pipettor

Take note: Never rotate the volume adjustor beyond the upper or lower

range of the pipette man, as stated by the manufacturer. Never use the pipette man without the tip in place; this could

ruin the precision piston that measures the volume of fluid. Never lay down the pipette man with filled tip; fluid could run

back into the piston. Never let plunger snap back after withdrawing or ejecting fluid;

this could damage the piston. Never immerse the barrel of the pipette man in fluid. Never flame pipette man tips.

If you drop your pipette man, the precision piston system can be damaged; therefore, if your pipette man is dropped, be sure to check the pipetting accuracy has not been affected.

Recommended Volume Ranges:

Model p10: 0.5-10 μL, the number after the decimal point is in redModel p20: 1-20 μL, the number after the decimal point is in redModel p200: 20-200 μL, there is no decimal pointModel p1000: 200-1000 μL, the numbers after the decimal point are in black

Pipetting Directions – Method1. Set the desired volume by holding the pipette man body in one

hand and turning the volume adjuster knob until the correct volume shows on the digital indicator. Approach the desired volume by dialing downward from a larger setting.

2. Press tip onto shaft by a slight twisting motion.3. Depress the plunger to FIRST POSITIVE STOP. This part of the

stroke is the calibrated volume displayed on the digital micrometer.

4. Holding the pipette man vertically (never more than 20˚ from vertical), immerse the tip just below the level of the liquid.

5. Allow the pushbutton to return SLOWLY to the up position. Move the tip so that it stays slightly below the level of the liquid as you draw up.

6. Wait one to two seconds to ensure that the full volume of sample is drawn up into the tip.

7. Withdraw the tip from the sample liquid.8. To dispense the sample, place the tip end against the sidewall of

the receiving vessel and depress the plunger to the FIRST

77

STOP. Wait one to two seconds. Then depress the plunger to the SECOND STOP, expelling any residual liquid in the tip.

9. With the plunger fully depressed, withdraw the pipette man from the vessel carefully with the tip sliding along the wall of the vessel.

10. Let the plunger return slowly to the UP position. If an air bubble is observed, re-pipette the sample.

11. Pre-rinsing the tip with the liquid being pipetted is recommended. A significant film may be retained on the inside wall of the tip, resulting in an error. Since the film remains relatively constant in successive pipettings with the same tip, refilling the tip a second time and using this quantity as the sample may obtain good reproducible results.

12. Discard the tip by depressing the tip ejector button smartly in the appropriate waste container.

Appendix E: Pipette ExercisesPipette Exercise #1

Determine and record the pipettor best suited for each of the measurements listed below.Add the indicated amounts to labeled microfuge tubes. Use the matrix below as a checklist while adding solutions to each microfuge tube.

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Solution 1 Solution 2 Solution 3Tube A (green) 10 µL 25µL 0.0963 mlTube B (red) 2 µL 0.015 ml 183 µLTube C (blue) 598.6 µL 0.200 ml 201.4 µL

Determine the total volume being added to each of the tubes. To check that your measurements are accurate, set a pipettor to the final volume and carefully withdraw the solution from each tube. Is the tip just filled? If measurements are inaccurate, repeat the exercise to obtain a near-perfect result.

Pipette Exercise #2

Using the p1000 and the p200 pipettors, perform the following:Set the pipettor to its maximum volume.Using water at room temperature, carefully pipette the water onto a weigh boat that you have tared (re-zeroed).Room temperature water has a density of approximately 1 gm/ml or 1 g/L. Therefore, you can determine the accuracy of your pipetting, e.g., 1000 L of water will weigh 1gm. Repeat the pipetting until you feel that you are reasonably accurate. Then record the weights of five successive pipettings. Determine the mean and standard deviation associated with your measurements.

Complete the following conversions:

1L =_______ml 0.001L =________ml10L =_______ml 0.11L =________ml100L =_______ml 0.01ml =________ml1000L =_______ml 1L =________ml

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APPENDIX F: GST Plasmid Map

APPENDIX G: Frequently Used DNA/Protein Markers

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Lambda DNA-Hind III Digest Prestained Protein Marker

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100 bp DNA Ladder 1Kb DNA LadderAPPENDIX H: Streak Plate Method

1 2

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3 4

5

Flame loop in between each step except between 4 and 5.Do not flame loop between steps 4 and 5.

APPENDIX I: PCR Reagents and Conditions for 1.17

Cycling Program: GST

94C 5 min Initial Elongation

94C 1 min

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5X50C 1 min72C 1 min

94C 1 min25X51C 1 min

72C 1 min

72C 10 min Final elongation

4C HOLD ***************

SAMPLESInitial Stock Concentration

Components 1 2 3 4 5 6 7

 Template DNA* 1-10 1-10 1-10

3 coloni

es

3 coloni

es 1-10 1-10  10X Buffer 5 5 5 5 5 5 525mM MgCl2 3 6 9 3 3 3 31.25mM dNTPs 8 8 8 8 8 8 8

20uMForward Primer 1 1 1 1 1 XXX 1

20uMReverse Primer 1 1 1 1 1 1 XXX

5U/ulTaq Polymerase 0.5 0.5 0.5 0.5 0.5 0.5 0.5

  dH2O              

*NOTE: Samples 1-3, 6 & 7: Use PLASMID DNASample 4: Transformed ColoniesSample 5: Non-Transformed ColoniesDESIRED FINAL VOLUME: 50µL

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APPENDIX J: PIERCE PROTEIN ASSAY for Module 2OD Value

Label L Alb (Stock 2mg/ml)

L dH2O

ml Dye

[Alb g/ml]

SeriesA

Series B

S1 0 100 2 0S2 2.5 97.5 2 50S3 5.0 95.0 2 100S4 7.5 92.5 2 150S5 10.0 90.0 2 200S6 12.5 87.5 2 250S7 15.0 85.0 2 300

LabelL

Sample

L d

H2Oml Dye OD Values g/ml in

Cuvetteg/ml

Original Solution

22222222

Label Average g/ml Original Solution of each treatment

g /lOriginal solution

l of protein that equates to 60g total

TransformedTransformed + IPTGControlControl +IPTG

APPENDIX K: Protein Gel Setup

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APPENDIX L: PIERCE PROTEIN ASSAY for Module 4OD Value

Label L Alb (Stock 2mg/ml)

L dH2O

ml Dye

[Alb g/ml]

SeriesA

Series B

S1 0 100 2 0S2 2.5 97.5 2 50

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S3 5.0 95.0 2 100S4 7.5 92.5 2 150S5 10.0 90.0 2 200S6 12.5 87.5 2 250S7 15.0 85.0 2 300

LabelL

Sample

L d

H2Oml Dye OD Values g/ml in

Cuvetteg/ml

Original Solution

2222

Label Average g/ml Original Solution of each treatment

g /lOriginal solution

l of protein that equates to 60g total

Treatment 1Treatment 2

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