DNA-practical of extracting
DNA
Aim
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
GATGGTGGTCAAGGGAC-3'
• Ready-prepared PCR mix, containing buffered dNTPs, Taq polymerase and MgCl2 (e.g., an Amersham-Pharmacia
‘Ready-togo’ bead)
• 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
• Stopclock
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 www.bioscience-explained.org 1 COPYRIGHT © BIOSCIENCE EXPLAINED, 2002 bioscience | explained Vol 1 | No 2
• Tips for
Aim
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
GATGGTGGTCAAGGGAC-3'
• Ready-prepared PCR mix, containing buffered dNTPs, Taq polymerase and MgCl2 (e.g., an Amersham-Pharmacia
‘Ready-togo’ bead)
• 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
• Stopclock
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 www.bioscience-explained.org 1 COPYRIGHT © BIOSCIENCE EXPLAINED, 2002 bioscience | explained Vol 1 | No 2
• Tips for