Download Comparing PCR of a Single Loci to Restriction Digest of the Entire

Human Cheek Cell DNA Extraction
1. Vigorously swish 5 mL of 0.9% salt solution (NaCl) in your mouth for 30 seconds. Chew on
your cheeks while swishing so you get as many cells as possible. Remember: more cells = more
DNA!!! Spit the salt solution back into your cup. Do this two times.
2. Mark a 2 ml tube on the lid with your initials and add 2 ml of your spit this tub.
3. Centrifuge the 2 ml tubes on high for 1 minute to collect cells at the bottom of the tube. Discard
the liquid, and add another 2 ml of your spit to the tube, repeat 5 times total.
4. Add 1.0 mL of Lysis Buffer to the cell pellet. Mix by carefully flicking the tube.
5. Add 50 l of Proteinase K (10 mg/ml). Flick the tube to break up the cell pellet.
6. Incubate the cells at 65-70°C for at least one hour, preferably overnight. Before proceeding, the
solution should be clear and the cells should not be visible.
7. Add 0.5 mL of 5M NaCl. Mix well, then centrifuge for 10 minutes on high speed.
8. Transfer the liquid equally (~500 l) into 3 new 2 mL tube. Do not bring the solids on the
bottom into the new tube, bring only liquid.
9. Add 1.5 mL of cold 95% ethanol to the liquid in each new tube.
10. Mix well by rocking the tube gently back and forth until the DNA becomes visible. It should
look like fine white fibers or lint.
11. Microcentrifuge on high for 5 min to pellet the DNA, discard the ethanol.
12. Let pellet dry completely (15-20 min at 37°C), add 100 l of TE or water to resuspend the
pellet in one tube, then bring that 100 l to tube 2 and resuspend the DNA into the liquid and final
bring the 100 l to tube 3 and resuspend the DNA into that liquid. In the end you should have all
of your DNA in 100 l of liquid.
You are now ready for PCR amplification of your own DNA or restriction enzyme digestion…..
Restriction Digest of Your DNA
The DNA double helix is made up of two strands of DNA held together by weak hydrogen bonds.
Since there are so many H-bonds the double helix is fairly stable, but the bonds can be easily
broken with heat or pH. The individual strands of DNA are held together with very strong
covalent bonds. The breaking of these bonds requires enzymes. Restriction enzymes are DNA
cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the
molecule, they are often called restriction endonucleases. The restriction enzymes we will be
using recognize very specific sequences in the DNA and then cut the DNA to produce fragments
called restriction fragments.
1. Label two microfuge tubes on the top of the lid and on the frosty side of the tube, one with
your initials and the enzyme you are using Hind III, the other with your initials and control
2. Using a micropipette with a clean tip each time, combine the following solutions into the
bottom of



3 l 10X buffer
l HindIII Enzyme
24 l of Genomic DNA
Close the tubes and centrifuge briefly (10 sec) to pool all of the liquid at the bottom of the tube
(if you do not have a centrifuge, tap or flick the tube contents to the bottom of the tube).
3. For the control DNA digest you will not add enzyme. Use a micropipette with a clean tip
each time, combine the following solutions into the bottom of a microcentrifuge tube:


6 l sterile water
l Extracted DNA
Close the tubes and centrifuge briefly (10 sec) to pool all of the liquid at the bottom of the tube
(if you do not have a centrifuge, tap or flick the tube contents to the bottom of the tube).
Place the microfuge tubes into a 37°C incubator overnight.
PCR Amplify Your DNA
Label a 0.2 ml PCR tube with your initials. Using a clean tip each time add the following to the
bottom of your PCR tube:





7 l water
10 l GoTaq
1 l Forward Primer
1 l Reverse Primer
1 l Extracted DNA
Close the tubes and centrifuge briefly (10 sec) to pool all of the liquid at the bottom of the tube (if
you do not have a centrifuge, tap or flick the tube contents to the bottom of the tube).
Set tube into thermal cycler and run HVRMTDNA
Initial denature 94°C 3 min
Then cycle 35 X
94°C 30 sec
56°C 1 min
72°C 2 min
Final extension 72°C 3 min and 4°C hold
Analyzing Your Restriction Digest and PCR Data
Materials:
DNA from restriction enzyme digests (BE SURE TO ADD 2 MICROLITERS LOADING
DYE!!)
DNA from PCR product (You do not need to add load dye, it is included in the GoTaq)
agarose
Tris-acetate/EDTA solution (TAE)
micropipette/tips
electrophoresis apparatus
Molecular Weight DNA Ladder (1Kb Plus)
Procedure:
1. Get your electrophoresis apparatus and make sure the stoppers are placed at either end of the gel
casting tray.
2. Pour hot agarose into the gel space until it reaches the top of the gel box. Make sure to place the
comb near the black electrode. Why? Let the agarose harden, which should take about 10 minutes.
Don’t touch/move your gel until it’s hard. Why not?
3. When the agarose gel is hard, take out the stoppers and pour TAE solution over your gel so that
is it completely covered plus a little more. What do you think the TAE solution is for?
4. Remove your comb and load 30 your restriction digest, PCR product and Molecular Weight
Marker into three of the wells near the BLACK ELECTRODE. Why near the black electrode?
Be sure to keep track of which samples you loaded in which lanes. Some one else can load their
restriction digest and PCR product into this gel too, you will only need one Molecular Weight
Marker per gel.
6. After you have loaded and labeled your samples in the gel picture below, you can go ahead and
run that gel!! Plug the electrodes into your gel box (red to red, black to black), being careful not
to bump your gel too much. Plug the power source set at 100-125 V into an outlet. How can you
tell your gel is running?
After about 30 minutes the DNA should be sufficiently separated to analyze, the dyes will have
migrated approximately 5-6 cm into the gel, turn off the power and carefully remove the gel. The
gel is very fragile, take care to not break it. You can remove the tray that you poured agarose on to
and gently slide the gel into the staining tray. At this point you cannot see the DNA, what can you
see and how do the different lanes compare? What can you do to see the gel?
Staining gels with Ethidium Bromide:
1. Put on gloves, you will be using Ethidium Bromide and will need to use care to not get it
on you. Ethidium Bromide is a known mutagen, and a possible carcinogen.
2. Place gel in staining tray
3. Remove the plastic from the ethidium bromide sheet and place the ethidium bromide paper
on the gel. Gently rub the paper with your fingers to make sure it is contacting the gel all
over.
4. Stain for about 10 minutes.
5. Put the gel on the UV light box and, with the UV shield down, view your gel.
6. Photodocument your gel and save the picture for your lab book and be sure to label the
picture.
7. Label the picture so that you will remember what sample is in which lane.
Questions:
1) What happened when you ran your uncut DNA on the agarose gel? Why do you think this
happened?
2) Did the DNA look different after you cut the DNA with a restriction enzyme? Did you expect
it to look different? Why or why not? How did the results reflect your expectations?
3) What did you see for the DNA you PCR amplified? Why do you think this happened?
4) Compare what was added to each of your DNA samples and explain what you think it will do to
your DNA:
Sample
DNA
Your
DNA
Water
Sample
Why
DNA Cut
add/what with
will
Enzyme
happen to
DNA?
Your DNA
Water
Buffer
Restriction
Enzyme
Sample
Why
PCR
add/what amplified
will
DNA
happen to
DNA?
Your
DNA
Water
Primers
Go Taq
Sample
Why
MW
add/what marker
will
happen to
DNA?
DNA
ladder
Water
Load
Dye
Why add/what
will happen to
DNA?
DNA different
sizes
Add volume
Dye for
electrophoresis