Difference between revisions of "TAS2R38 PCR amplification"
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'''PCR conditions:''' | '''PCR conditions:''' | ||
# 5 minutes at 95˚C. | # 5 minutes at 95˚C. | ||
| − | # | + | # 30 seconds at 95˚C. |
| − | # | + | # 30 seconds at 55˚C. |
| − | # | + | # 30 seconds at 72˚C. |
# repeat steps 2 - 5 29 more times. | # repeat steps 2 - 5 29 more times. | ||
# hold at room temperature. | # hold at room temperature. | ||
Revision as of 20:30, 13 August 2012
1. When the DNA extraction cools (Isolate_genomic_DNA_from_a_single_hair_follicle), vortex the tubes for 30 seconds and then set up a new 500 µL microfuge tube by adding the following:
| Reagent | Volume |
|---|---|
| extracted DNA | 10 µL |
| dH2O | 13 µL |
| 2X Green GoTaq Mix | 25 µL |
| Forward Primer | 1.0 µL of 10 µM stock |
| Reverse Primer | 1.0 µL of 10 µM stock |
| Final Volume | 50.0 µL |
2. Run the thermocycler program called TAS2R38 with the heated lid enabled. PCR conditions:
- 5 minutes at 95˚C.
- 30 seconds at 95˚C.
- 30 seconds at 55˚C.
- 30 seconds at 72˚C.
- repeat steps 2 - 5 29 more times.
- hold at room temperature.
- END
3. When the PCR is completed (~2 hours), store DNA frozen until you are ready to clean up the DNA in order to have your two alleles sequenced.
4. Before you can send your DNA off for sequencing, you will need to clean up your PCR products
Reference:
