Lab Methods in Molecular BiologyBio 446-546

FALL 2015 M/F 13.00-15.50

Molecular biology of parasites, snails, plants and fungi

You, Adema, Natvig, Hutchinsonhttp://biology.unm.edu/cmadema/4546/4546.htm

TodayIntroductions

Goals and expectations for this course

Safety video

Organization

Background for research project

Introduction to pipetting

Pipetting exercise

Troubleshooting questions

IntroductionDonald (Don) O Natvig

Professor

(Fungal Genetics)

Coenraad (Coen) M Adema

Associate Professor

(Comparative Immunology/Parasitology)

Miriam HutchisonPhD Graduate Student

(Fungal Genetics)

NOT “MOLECULAR BIOLOGISTS”, USE RESEARCH TOOLSNOT “MOLECULAR BIOLOGISTS”, USE RESEARCH TOOLS

WHO ARE YOU?WHO ARE YOU?

Molecular Biology• Tools for biological study• Expensive• Difficult (despite X-files and CSI)• Frustrating• Complex theory hidden in kits, industry• Slow

• Getting bigger BIGGER BIGGER• Computer intensive BIOINFORMATICS• Great fun

Goals and expectations for this coursePhilosophy and major objective of the Course: It is easy to follow a recipe, it is

harder to follow it well, and most difficult to make it work. Our major objective is to teach you to get things to work in the lab, this may not be difficult for some of you, but it may require a personality change for others.

There are 5 identifiable goals in this course:(1) To introduce you to modern techniques used in molecular biology research.(2) To teach you how to obtain and apply computer- and internet-based resources for

molecular biology(3) To teach you to perform research carefully so that your scientific observations

stand on solid ground. (4) To troubleshoot experiments, but most of all, to persevere when nothing works.(5) To give you experience in communicating research findings to the scientific

community.

Ideally, this course will give you a better understanding of what a scientific career that uses molecular techniques would be like and inspire you to pursue additional studies.

Syllabus ONLINE

Course information such as lectures, suggested reading, handouts, and homework assignments will be posted online at: http://biology.unm.edu/cmadema/4546/4546.htm

Textbook: NOTE there is NO mandatory book for this class. Readings will be provided through the above website in the form of protocols, scientific papers, also check out http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books

For those that prefer the option of a textbook for background information, we recommend: Molecular Biology of the Gene (7th ed), Watson, Baker, Bell, Gann, Levine, Losick, Publised by Pearson student edition ISBN 13:978-0-321-76243-6, ISBN 10: 0-321-76243-6

Grading IGrading (400 points undergrad/500 points for grad students)

Your final grade will depend on:• midterm examination (take home) (100)• 15 minute class presentation (50)• your lab book (25)• Homework/in class troubleshooting exercises (50) • class attendance/participation (75)• final paper in journal submission manuscript format (100). • Additionally, graduate students will be required to give the class a 30 minute presentation on their (proposed) research (100).

• Unexcused/excessive absences will lower your final grade. Do not make it a habit of arriving to class late. The class depends on you to be here so that we can start and finish class on time.

• FYI-you will work in groups of two/three throughout the semester. You are part of a team, so don’t let your partners down. Do your share of the work, be respectful of each other’s grasp of the material, help each other whenever possible. As is common in science, you will collaborate on the writing on your final paper and turn one paper in per group.

• Warning: There are always slow groups in the class. Please be respectful. It is our experience that these are often the groups that get the best results..

• Grade Mode Changes: The WP, WF, and WNC are no longer available since Fall semester 2012.  Instead, we have a W (withdrawal) grade mode that does not impact student GPA. (Remember that the course, if dropped after relevant deadlines remains on the student record and in their tuition account)  Like before, this grade mode does require a Last Date of Attendance (LDA).  Please see Fastinfo 3489. https://unm.custhelp.com/app/answers/detail/a_id/3489/kw/grade%20mode/session/L3RpbWUvMTM0NDg4MDA2Mi9zaWQvS21FTS1CM2w%3D

Grading IILab Notebook (25 points):

• You are required to keep a lab notebook for this class. This should be a running commentary with your ideas and notes to yourself, together with all of the raw data and any calculations you make from the raw data. Information about the equipment you use should be listed as should information about the chemicals you use. It should be possible for someone generally familiar with your area of study to pick up your notebook and repeat or continue your experiments.

• All of the exercises will require making calculations or summarizing your results in a notebook. This should be done during the scheduled lab period for two reasons.

– First, it is usually easier to do calculations when the data is fresh in your mind and you are more accurate in recalling details for your summaries when you write them immediately.

– Second, the instructor will be available during the lab period and may not be available later. One of the criteria on which you will be graded is how current you notebook is. Lab notebooks will be checked by us periodically.

• Evaluation: You will be evaluated on the basis of the laboratory notebook you will keep. You should record everything of interest to you in the lab in this notebook. It is not necessary to reproduce methods spelled out in the handouts, but everything else should be in your notebook. Not everything has to be polished, for example, if you want to do a small calculation, do it in your notebook, not on a paper towel (formerly the record keeping choice of most scientists). Within the last few years society has given clear signals that it expects scientists to keep reasonable records and we must all learn to do this. All written communication in the lab will be in this notebook, including feedback from the instructors. Do not write your raw data on paper towels intending to transfer it to the notebook later. If you want to have a neat record of your results and feel you cannot do this as the experiment progresses, take the raw data on one page, and then copy it to a later page in the notebook. You will not be graded on neatness.

Notebook keepingLab Notebook:

General Guidelines:

• 1. Use a bound book of decent size with numbered pages (hand numbering is okay).• 2. Make all entries in ink, beginning with your name and some identification about the notebook.• 3. Leave 5 pages or more in the front for a table of contents. For each experiment or set of closely related experiments,

make an entry in the table of contents followed by the date of the experimental work, and the page number where the research is described in detail.

• 4. Each experiment should list the – Title and date– One or two sentences about the experiment– Methods (including calculations and materials)– Summary of the results (generally with some sort of graph or diagram)– Short conclusion stating important findings and/or problems encountered.

• 5. Changes on previous pages, when needed, should be made by crossing out the old information with one line and putting the new information nearby. Date any changes made to your notebook.

• 6. Raw data such as photos of your gels can be cut out and taped into the notebook. When particularly voluminous raw data is involved, it is best to store this separately, but with a good cross reference system so that the data and the description of the data can be put together later.

• 7. Rough graphs can be drawn directly in the notebook; more formal graphs should be drawn on appropriate graph paper or with a graphing program, and taped into the notebook.

Safety video

Working in the lab• Laboratory Rules and Guidelines:

– Be safe working with dangerous equipment and chemicals,– Ask the instructors when you have questions.

• Laboratory etiquette (for your safety):– No food (this includes chewing gum) or drink is allowed in the lab. – Cell phones, music players and other electronic devices should be turned off.– Books (other than your notebook and lab manual), backpacks, clothing, and personal belongings should not be

kept on lab benches.– Lab coats and protective eyewear are recommended when conducting experiments. Lab gloves should be worn

whenever working with chemicals or solutions. Do not wear shoes with open toes in the lab.– Handle all equipment carefully and don’t goof around in the lab.– Wash and rinse all glassware when you finish using it.– Return individual items to your drawer at the end of each lab.– Notify the instructor if any spills occur or if equipment is damaged, and assist as instructed for the clean-up.– Notify the instructor if anything is spilled on your lab notebook or manual. It is important not to take contaminated

material out of the lab.

• Waste disposal:– In biohazard bags (tips, tubes, plates, gels)

• Sharps disposal:– Glass and sharp objects should be disposed of in special containers found around the lab. Never, under any

circumstances, put sharp things in the regular trash. This presents a very real hazard for maintenance workers.

SCHISTOSOMIASIS:A GLOBAL PROBLEM

•207 million humans infected •Annual mortality 280,000 •Morbidity 120 million humans

•600 million more humans at risk

•Disease impact of schistosomiasis second only to malaria (WHO)

•Effective chemotherapy, praziquantel currently used in mass treatment program targeting morbidity by the Schistosomiasis Control Initiative.

•Concern for longevity of parasites, parasite resistance, un-sustainability of ongoing mass treatment efforts, absence of new drugs and vaccine.

•Continued study of all aspects of schistosomiasis remains valid,

cercariae penetrate

human skin

adult wormsdevelop inmesenteric

veins

spined eggs

passed inthe feces

miracidiumhatches

from egg in freshwater,

penetrates snail

asexual reproductionoccurs in freshwatersnails of the genus

Biomphalaria

cercariaeproduced

in the snail digestive

gland

cercariae released

into water

THE LIFE CYCLE OF THE LIFE CYCLE OF Schistosoma mansoniSchistosoma mansoniMALE

FEMALE

http://www.dpd.cdc.gov/DPDx/Default.htm

http://www.biologie.uni-erlangen.de/parasit/contents/research/echino.html

greenmuseum.org/c/vban/trematode.phphttp://parasitology.informatik.uni-wuerzburg.de/login/n/h/0103.html

Apophallus muehlingi

Alaria americanum

cal.vet.upenn.edu/merial/Trems/fascio_c.htm

Shady lakes facebook

PARASITES AND SNAIL BIOLOGY

“identity, possibilities”phylogenetics

“intentions”transcriptomics

PCRrDNA/mito

BioanalyzerDNA-free,

direct sequencing

gel electrophoresisnanodrop spec

Sequence ID (BLAST)editing

Phylogenetics

electrophoresisRT-PCR

gel

CTAB/DNAzol

Trizol

TA cloning, B/W screening

M13 sequencing

Primer design, walking

Qiagen plasmid extraction Restriction digests

DNA

RNA

GenBank submission

groups

• You will work in groups of two or three• Graduate students team up with

UnderGraduates• groups will be responsible for

– Experimental work– Final report using experimental data

Time to go through your drawers

• drawer inventory should match what is in your drawers

• Mark off items and sign form

• Return to instructors

• We will repeat this on the last day of class

• Name + picture

P20: 2(1)-20 µl = .002-.020 ml (use “yellow” tips)10 = 10.2 µl2 P200: 20-200 µl = .020-.200 ml (use “yellow" tips)10 = 102 µl2 P1000: 100-1000 µl = .100-1.0 ml (“blue tips”)10 = 1020 µl (1.02 ml)2

Pipettors, volume ranges

Pipetting demonstration• Price of pipettor• Volume• Range, adjust volume • Tips• Double jam, twist (hold the box!)• First stop, pre-wet, view volume, repeat• Dispense: second stop, look for drop or bubble• Mix: down, up and down to first stop • Move tip out of liquid• Eject

Pipetting exercise

• LEARN HOW TO PIPETTE• Tips• Hold the pipettor vertically• Use a smooth up and down motion• Pre-wetting your tip improves accuracy