The Analysis of Pre-Digested Lambda () DNA and

Determining the lengths of each fragment Produced. using Gel

Electrophoresis

Lab #7

(Chapter 20)

Materials for Lab• Materials List @ each Station:

– (5) Vials w/ appropriate contents (see quick guide)– 500 ml Beaker: used for TAE Buffer (need 300ml)– Case of pipette tips---be careful with!!!!– Electrophoresis Chamber– Pre-casted Gel with six wells prepared– Micropipette (to transfer 10 L)– 100 ml Grad. Cylinder– One Staining tray– Tape & sharpie pen

• Other Supplies Needed located at other places:– 1x TAE Buffer Solution ------------------------------- on the cart– 1x Fast Blast Stain (need 120 ml)------------------- on the cart– Power Supplies: 2 in the back & one in the front of room

Basic Procedure I• Look over your supplies and lay them out• Locate the pipette tips and micropipette and practice moving

10 µL form blue “LD” tube to the clear tube (in beaker)• Obtain 300ml of TAE Buffer solution from the back of room

(use your Beaker) & pour into the chamber.• Look at the Quick guide and ID the contents in the colored

tubes.• Skip to step 3 & 4. Remove a gel cast from the sealed

baggie. Remove the tape from both ends carefully. • Make a small corner cut on one end at the top of the gel near

the six wells. Leave the gel on the tray.• Place the gel & tray into the chamber. Be sure that the wells

are near the black (-) electrode. • Pour the 1x buffer solution into the chamber. Do not pour

directly on to the gel.• Gently push down on the tray to submerge the entire gel and

tray. The little holes will allow any air under the tray to escape allowing it to sink.

Loading the Gels w/Micropipette• Be sure that your Micropipette is set at “10.0”• Load your DNA samples into the correct well as

follows from left to right using a micropipette.Closet to the corner cut of the gel.

– Well #1: Lambda DNA (uncut) (yellow tube)– Well #2: Lambda cut w/PstI (violet or pink)– Well #3: Lambda cut w/EcoRI (green tube)– Well #4: Lambda cut w/Hind III (orange tube)

I will take two groups at a time in the back or front of the room. While you wait answer all questions from your guide sheet. Pgs. 21-28

Basic Procedure III

• Once all wells are loaded, Cover your chamber with the lid.

• Attach your electrodes to the power source. Black = Negative

Red = Positive

Current can be: 100 V for 30 min. ideally

45 V for 3 hrs.

Staining your Gels

For Overnight Staining:

-Pour 120 ml of Staining solution (1X Fast Blast) into the tray. Let gels stand overnight. Pour off fluid next day. Record your results.

**To record you results bring either a camera or you may draw it.

Steps Outlined in your “Quick Guide”

Adding each digest to the correct well

• Well #1 (Lane) = uncut Lambda DNA “L”

• Well #2: Lambda DNA cut w/ PstI “P”

• Well #3: Lambda DNA cut w/ Eco RI“E”

• Well #4: Lambda DNA cut w/ HindIII“H”

After Gel is done Running

• We will do Option 2 for visualizing our DNA “Overnight staining”

• Fill your staining tray w/ 120ml of Fast Blast stain

• Let stand overnight-cover w/saran wrap

• Next day: pour off water

• Record your results: Draw, pictures, etc.

• Let dry and sit on your casting tray

Introductory Questions #4Using your Lab Guide and textbook answer the following

questions:1) Where do we obtain Restriction enzymes

(endonucleases) and how are they named? 2) What name do we use to describe two sequences of

DNA that read the same in both directions? (lab pg. 22) Why are these sequences important?

3) If a restriction enzyme made 9 cuts on the DNA then how many fragments would there be?

4) To separate fragments of DNA Gel Electrophoresis is used. What voltage did we use and for how long? Which side (+/-) of the gel was the DNA loaded?

5) How many base pairs does lambda DNA have? (see pg. 29)

Restriction Enzymes (pg. 386)

Restriction Enzymes (Endonucleases)

• Found in Bacteria & named as such: Restriction enzyme Name of Bacteria

– EcoRI Escherichia coli– Hind III Haemophilus influenza– Pst I Providencia stuartii– Bam I Bacillus amyloliquefaciens

• Recognizes Palindromic Sequences• Cuts the all DNA and produces pieces (fragments)

called RFLP’s. (Pg. 394)• RFLP: Restriction Fragment length Polymorphisms• RFLP’s can measure genetic relationships and

generate a DNA fingerprint when separated on an agarose gel.

Action of Restriction Enzymes-Pg. 386

Restriction Fragment Analysis• Restriction fragment length polymorphisms (RFLPs)• Southern blotting: process that reveals sequences

and the RFLPs in a DNA sequence• DNA Fingerprinting (DNA Profiling)

Gel Electrophoresis

• DNA fragments placed into “wells” in gel agarose

• Electricity pulls on DNA fragments• Fragments travel at

different rates based on size and ability to squeeze through swiss-cheese-like agarose

Setting up a Gel Electrophoresis Unit

Gel Electrophoresis Pgs 392-394• separates nucleic acids or proteins on the basis of size and

electrical charge creating DNA bands of the same length

• DNA has a net negative charge (use a positive charge in the gel)

Lamda DNA-used in this lab• DNA comes from a Bacteriophage

• Genome composed of 48,502 base pairs

• Known: -(7) restriction sites for Hind III-Sizes for all eight Fragments

Restriction Enzymes used in the LabName of Enzyme Sequence Recognized

“Palindrome”

-EcoRI G ….A A T T C

C T T A A…. G

-PstI C T G C A G G A C G T C

-HindIII A A G C T T

T T C G A A

Introductory Questions #4Using your Lab Guide and textbook answer the following

questions:1) Where do we obtain Restriction enzymes

(endonucleases) and how are they named? 2) What name do we use to describe two sequences of

DNA that read the same in both directions? (lab pg. 22) Why are these sequences important?

3) If a restriction enzyme made 9 cuts on the DNA then how many fragments would there be?

4) To separate fragments of DNA Gel Electrophoresis is used. What voltage did we use and for how long? Which side (+/-) of the gel was the DNA loaded?

5) How many base pairs does lambda DNA have? (see pg. 29)

Intro. Q’s #5 for Chapter 20: Genetic Engineering1) What does the acronym PCR stand for and what does this

process do?2) What does Gel electrophoresis allow us to do?3) Give two applications of DNA profiling.4) What are the advantages and disadvantages of genetic

screening?5) How is a cDNA library different from a genomic library? (p.

388-390)6) Name two “vectors” that can be used for gene transfer.7) Give two examples of a genetically modified crop or animal. 8) Briefly explain the process of gene therapy and give an

example how it works.9) Explain what a clone is and how it could be formed.10) What are some of the ethical concerns about cloning? Give

your opinion if you think cloning is something we should be doing.

Seven Restriction sites (8 fragments) using Hind III

Pg. 29

Analyzing Your Data Determine the approximate size of your

fragments using the known Hind III digest patterns as a standard (marker).

1) Visual estimation2) Create a standard curve3) Measure the distance (mm) each band traveled

from the well. Be sure to measure from the bottom of the well to the bottom the band.

4) Record your measurements in the table5) Estimate (visually) the number of base pairs the

fragments would have by comparing with the actual number of base pairs for Hind III Fragments

Sample Data (Graph data on pg 74)

Constructing a Standard Curve

• Graph the following on Semi-log paper– (y-axis): Size of known fragments (Base pairs)– (x-axis): Distance each band moved (mm)

****Note: HindIII is used

• Use a ruler and for each band measured, line up the distance it traveled with the standard line drawn. Look horizontally on the y-axis and determine the the number of Base pairs those fragments should be.

2nd Table will be needed for your data or the data from the image on ppt slide

Print out & Paste on pg. 34 of your lab if your bands are not visible

DNA DNA DNA DNA

Uncut w/PstI w/EcoRI HindIII

Measure (mm)

Fragment #1

Fragment #2

Fragment #3

Visible Fragments

Semi-Log Graphing Paper Pg. 40Graph your data or data from ppt slide

Key Points to Remember • The same DNA was used with all three enzymes • We know the Fragment sizes using HindIII (p.29)

– Seven cuts & Eight Fragments

• Will compare the fragments generated from PstI & EcoRI to that of Hind III in order to estimate how long the fragments fragments are.

• This comparison will be observed through the separation of these fragments on Gel agarose.

• Four wells will be used to compare

Lab #7: Homework

• Answer the 3 bulleted Questions on Pg. 42

• Respond to Questions on pg. 75-77.

• Lab is Due on Thurs. 12/10

Biogenetic Engineering & Manipulating Genes

Chapter 20

Genetic Engineering

• Chapter 20• DNA Technology &

Genomics

Pgs. 384-396 & 402-408

O.J. Simpson capital murder case,1/95-9/95• Odds of blood in Ford Bronco not being R. Goldman’s:

• 6.5 billion to 1• Odds of blood on socks in bedroom not being N. Brown-Simpson’s:

• 8.5 billion to 1• Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:

• 21.5 billion to 1• Number of people on planet earth:

• 6.1 billion• Odds of being struck by lightning in the U.S.:

• 2.8 million to 1• Odds of winning the Illinois Big Game lottery:

• 76 million to 1 • Odds of getting killed driving to the gas station to buy a lottery ticket

• 4.5 million to 1• Odds of seeing 3 albino deer at the same time:

• 85 million to 1• Odds of having quintuplets:

• 85 million to 1• Odds of being struck by a meteorite:

• 10 trillion to 1

Tools of Genetic Engineering• Restriction enzymes (endonucleases)

-in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific

• Restriction site:

-recognition sequence for a particular restriction enzyme• Restriction fragments:

-segments of DNA cut by restriction enzymes in a reproducable way

• Sticky end:

-short extensions of restriction fragments• DNA ligase:

-enzyme that can join the sticky ends of DNA fragments• Cloning vector:

-DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

DNA Fingerprinting

• Band Patterns observed on a gel when the restriction fragments are separated

• Used in:– Crime scenes– Determining Paternity– Finding Evolutionary trends & commonalities– Gene therapy– Diagnosing genetic disorders– Generating Transgenic Plants: feeding people

Polymerase Chain Reaction (Pg. 391-392)

• Discovered in 1985• Used to “amplify” or make many copies of DNA.• Important in forensic science• Involves:

– Heating separates the two strands– Adding DNA Polymerase (heat resistant)

(Thermus aquaticus-Taq) : found in Hot Springs @ Yellowstone

– Adding primers: short Single stranded DNA– Adding Nucleotides: dATP, dGTP, dTTP, dCTP– Cooling: allows for hydrogen bonds to form**Can form over a million copies in only 20 minutes

PCR-Polymerase Chain Reaction (Pg. 391-392)

PCR-Polymerase Chain Reaction

Steps for Eukaryotic gene cloning

Cloning Genes using Bacterial Plasmids

Results After Staining

Lab #2-Results

Hind III

Crime

Scene

Suspect #1 Suspect #2

Extra band

Similar pattern

Hind III EcoR I

Bottom of well

Bottom of each band (mm)

Measure the following