From 2011.igem.org
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Revision as of 16:15, 27 September 2011
Taq PCR Protocol
Reagent |
Volume ( 1x ) |
Volume ( 3x ) |
Volume ( 5x ) |
Volume ( 15x ) |
Sterile H2O |
36 μL |
108 μL |
180 μL |
<td>540 μL</td>
</tr>
<tr>
<td>10X Taq Buffer</td>
<td>5 μL</td>
<td>15 μL</td>
<td>25 μL</td>
<td>75 μL</td>
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<td>2mM dNTPs</td>
<td>5 μL</td>
<td>15 μL</td>
<td>25 μL</td>
<td>75 μL</td>
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<tr>
<td>Forward Primer (100 ug/ul) </td>
<td>1 μL</td>
<td>3 μL</td>
<td>5 μL</td>
<td>15 μL</td>
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<tr>
<td>Reverse Primer (100 ug/ul) </td>
<td>1 μL</td>
<td>3 μL</td>
<td>5 μL</td>
<td>15 μL</td>
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<td>50mM MgCl2</td>
<td>1.5 μL</td>
<td>4.5 μL</td>
<td>7.5 μL</td>
<td>22.5 μL</td>
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<td>Taq Polymerase (50 ug/ul)</td>
<td>0.5 μL</td>
<td>1.5 μL</td>
<td>2.5 μL</td>
<td>7.5 μL</td>
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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
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