Team:Calgary/Notebook/Protocols/Process9
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<p><b> Thermocycler Conditions </b></p> | <p><b> Thermocycler Conditions </b></p> | ||
<ol> | <ol> | ||
- | <li> 1 Cycle - 6 minutes at 95 | + | <li> 1 Cycle - 6 minutes at 95°C </li> |
<li> 36 cycles of: | <li> 36 cycles of: | ||
<ol> | <ol> | ||
- | <li type="a"> 1 minute at 95 | + | <li type="a"> 1 minute at 95°C </li> |
- | <li type="a"> 1 minute at 58 | + | <li type="a"> 1 minute at 58°C (this step done at 65°C for higher GC content ) </li> |
- | <li type="a"> 1 minute at 72 | + | <li type="a"> 1 minute at 72°C </li> |
</ol> | </ol> | ||
</li> | </li> | ||
- | <li> 1 Cycle - 10 minutes at 72 | + | <li> 1 Cycle - 10 minutes at 72°C then HOLD at 4°C</li> |
</ol> | </ol> | ||
<p> 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</p> | <p> 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</p> |
Latest revision as of 04:26, 29 September 2011
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Taq PCR Protocol
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The following is obtained from the Invitrogen Taq PCR kit (although most PCR kits have similar protocols).
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°C
- 36 cycles of:
- 1 minute at 95°C
- 1 minute at 58°C (this step done at 65°C for higher GC content )
- 1 minute at 72°C
- 1 Cycle - 10 minutes at 72°C then HOLD at 4°C
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.
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