This practical procedure allows you to amplify a 460 basepair fragment of DNA from within the control region of the
mitochondrial genome. This can be done using three water baths or, if one is available, a thermal cycler (PCR machine). After it has been amplified, the DNA is run on an electrophoresis gel.
Note: This method has been adapted from one developed by the Dolan DNA Learning Center at Cold Spring Harbor Laboratory. More details are available from the DNA Learning Center’s Web site: http://vector.cshl.org/geneticorigins
Equipment and materials
Needed by each person or group
For extracting the DNA, and the PCR
• Isotonic sports drink, 3–5 ml (orange-flavoured is useful, as it
stains the extracted cells so that they are easier to see)
• Clean, disposable plastic or paper cup
• Disposable plastic syringe (1 ml, without a needle)
• Chelex resin 500μl (10% in 50 mM Tri-buffert, pH 11)
• Primers, 5 μl of each (in Cresyl Red loading dye — See page 9):
5'-TTAACTCCACCATTAG CACC-3'5'- GAG
• Ready-prepared PCR mix, containing buffered dNTPs, Taq
polymerase and MgCl2 (e.g., an Amersham-Pharmacia
• Sterile distilled or deionised water, 10 μl
• Microcentrifuge tubes, 2 (1.5 ml)
• Mineral oil (only needed if you use a thermal cycler without a heated head plate)
• Micropipette or similar device, with a 5–40 μl range
• Tips for micropipette
• Thermal cycler or...
For manual cycling
• Water baths, 3 (maintained at 94, 55 and 72 °C)
• Floating holder for microcentrifuge tubes
For the electrophoresis
• Agarose solution, (1.5% in TBE buffer, melted in a
microwave oven then kept molten in a water bath or
incubator at 55–60 °C)
• TBE buffer
• Azure A solution for staining the gel (0.04% in 20% ethanol) • Micropipette or similar device, with a 5–40 μl range
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• Tips for micropipette
• Gel electrophoresis equipment and power supply
• OPTIONAL: 1 kb DNA ladder solution, 20 μl in TE buffer
(containing 1 μg of DNA)
or 20 μl of Lambda DNA (containing 2 μg of DNA) digested
with Hind III.
You will also need
• Marker pen, fine tipped, permanent
A Isolation of mitochondrial DNA
1 Rinse your mouth with the isotonic sports drink for 60–90 seconds. Use your teeth to scrape cells gently from the sides of your mouth as you do this. Cheek cells, containing
mitochondria, will be washed into the drink. As the drink is isotonic, the cells will be prevented from bursting open (lysing). 2 Spit the drink, with saliva and cheek cells into a disposable cup. 3 With a sterile syringe, measure 1 ml of the drink and cells into a microcentrifuge tube. Take care to suck up some of cells from the bottom of the cup, but try to leave any frothy saliva behind. 4 Spin the tube for 5 minutes at 13 000 rpm.
WARNING! Ensure that the centrifuge is balanced!
5 Tip the supernatant into a waste container of disinfectant. Do this smartly and confidently — the pellet of cells should stay in the tube. If you do not obtain a pellet at this stage, you have probably sucked up too much saliva and not enough cells. Repeat steps 3, 4 and 5 until you obtain a clearly-visible pellet.
6 Leave the tube to stand, inverted, on a paper tissue so that most of the liquid drains away. A small amount of the liquid will remain in the tube. Dispose of the paper in the disinfectant. Fig. 1 Fig. 2 Fig. 3
Fig. 4 Fig. 5 Fig. 6
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7 Use a syringe to add 0.5 ml of Chelex resin to the pellet. Make sure that the beads of resin are properly suspended in the buffer as you add them. Note: The resin beads do not dissolve in the liquid.
8 Cap the tube, then flick it firmly to resuspend the cells. This can be quite difficult to do, and you may have to persevere and shake the...
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