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Team:Calgary/Notebook/Protocols/taqpcr
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<p>The following is obtained from the Invitrogen Taq PCR kit (although most PCR kits have similar protocols).</p> | <p>The following is obtained from the Invitrogen Taq PCR kit (although most PCR kits have similar protocols).</p> | ||
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| - | + | <td width="85%"><div class="heading"><center><h3></a>Mastermix setup</h3> </center></p></div></td> | |
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| + | <p><i>Thermocycler Conditions</i></p> | ||
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| - | <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> |
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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> | ||
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| + | <p>We also used a Taq polymerase that we isolated in-house with slightly different cycling conditions. Since this Taq could not tolerate the lysis stage at 95°C for 10 minutes, the cells were lysed in 4 μL of water for 10 minutes before master mix was added. | ||
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Latest revision as of 03:54, 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 lysis stage at 95°C for 10 minutes, the cells were lysed in 4 μL of water for 10 minutes before master mix was added.
