Human Cheek Cell DNA Extraction
1. Vigorously swish 5ml 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. Repeat this process with new 5ml of salt solution.
2. Mark a 2ml tube on the lid with your initials and add 2ml of your spit to this tube.
3. Centrifuge the 2ml tubes on high for 1 minute to collect cells at the bottom of the tube. Be sure to balance your tube in the centrifuge! Discard the liquid, and add another 2ml of your spit to the tube, repeat 5 times total.
4. Add 1.0ml of Lysis Buffer to the cell pellet. Mix by carefully flicking the tube.
5. Add 50ml of Proteinase K (10mg/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 0.5ml of 5M NaCl. Mix well, then centrifuge for 10 minutes on high speed.
8. Transfer the liquid equally (~500ml) into 3 new 2mL tube. Do not bring the solids on the bottom into the new tube, bring only liquid.
9. Add 1.5ml of cold 95% ethanol to the liquid in each new tube.
10. Mix well by rocking the tube gently back and forth; the DNA may become visible. If it does, 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 42°C), add 100ml of TE or water to resuspend the pellet in one tube, then transfer that 100ml to tube 2 and resuspend the DNA into the liquid and finally transfer the 100ml to tube 3 and resuspend the DNA into that liquid, in the end you will have all of your DNA consolidate in one tube in 100ml of liquid.
You are now ready for PCR amplification of your own DNA.
Review PCR:
www.dnalc.org/resources/animations/pcr.html
With voice over on YouTube:
http://www.youtube.com/watch?v=JRAA4C2OPwg
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:
· 14ml water
· 20ml GoTaq
· 2ml Forward Primer
· 2ml Reverse Primer
· 2ml 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 the MT2 program
Initial denature 94°C 3 min
Then cycle 35 X
94°C 30 sec
54°C 45 min
72°C 30 min
Final extension 72°C 3 min and 4°C hold
Analyzing the PCR Reaction
Materials: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 15ml of your PCR product and 15ml of Molecular Weight Marker into 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 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 the 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 which sample is in which lane.