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Team:Calgary/Notebook/Protocols/Process9
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Revision as of 21:29, 27 September 2011
TITLE=Taq PCR Protocol| BODY=
| Reagent | Volume ( 1x ) | Volume ( 3x ) | Volume ( 5x ) | Volume ( 15x ) |
| Sterile H2O | 36 μL | 108 μL | 180 μL | 540 μL |
| 10X Taq Buffer | 5 μL | 15 μL | 25 μL | 75 μL |
| 2mM dNTPs | 5 μL | 15 μL | 25 μL | 75 μL |
| Forward Primer (100 ug/ul) | 1 μL | 3 μL | 5 μL | 15 μL |
| Reverse Primer (100 ug/ul) | 1 μL | 3 μL | 5 μL | 15 μL |
| 50mM MgCl2 | 1.5 μL | 4.5 μL | 7.5 μL | 22.5 μL |
| Taq Polymerase (50 ug/ul) | 0.5 μL | 1.5 μL | 2.5 μL | 7.5 μL |
Thermocycler Conditions
- 1 Cycle - 6 minutes at 95 degrees Celsius
- 36 cycles of:
- 1 minute at 95 degrees Celsius
- 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content )
- 1 minute at 72 degrees Celsius
- 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius
Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 or even 3 minutes
Follow the same protocol for a colony PCR.
