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Team:Calgary/Notebook/Protocols/taqpcr
From 2012.igem.org
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<p><i>Thermocycler Conditions</i></p> | <p><i>Thermocycler Conditions</i></p> | ||
<ol> | <ol> | ||
| - | <li> 1 Cycle - | + | <li> 1 Cycle - 10 minutes at 95°C </li> |
<li> 36 cycles of: | <li> 36 cycles of: | ||
<ol> | <ol> | ||
| - | <li type="a"> | + | <li type="a"> 3 minutes at 95°C </li> |
| - | <li type="a"> 1 minute at | + | <li type="a"> 1 minute at 55°C (for BioBrick primers) </li> |
| - | <li type="a"> 1 minute at 72°C </li> | + | <li type="a"> 1 minute/kb at 72°C </li> |
</ol> | </ol> | ||
</li> | </li> | ||
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<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> | ||
<p>Follow the same protocol for a colony PCR.</p> | <p>Follow the same protocol for a colony PCR.</p> | ||
| + | |||
| + | <p>We also used a Taq polymerase that we isolated in-house with slightly different cycling conditions. Since this Taq could not tolerate the 95 degree heat required for the 10 minutes the initial lysis step in colony PCR, the cells were lysed on their own for 10 minutes before master mix was added. | ||
</div> | </div> | ||
</td> | </td> | ||
Revision as of 03:40, 4 October 2012
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Taq PCR Protocol
The following is obtained from the Invitrogen Taq PCR kit (although most PCR kits have similar protocols).
Mastermix setup |
| 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 - 10 minutes at 95°C
- 36 cycles of:
- 3 minutes at 95°C
- 1 minute at 55°C (for BioBrick primers)
- 1 minute/kb 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.
We also used a Taq polymerase that we isolated in-house with slightly different cycling conditions. Since this Taq could not tolerate the 95 degree heat required for the 10 minutes the initial lysis step in colony PCR, the cells were lysed on their own for 10 minutes before master mix was added.
