Team:Calgary/Notebook/Protocols/taqpcr
From 2012.igem.org
(Difference between revisions)
Line 5: | Line 5: | ||
<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> | ||
- | + | ||
<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
<table width="700"> | <table width="700"> | ||
<tr> | <tr> | ||
- | <td width="85%"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="pcr"></a>Taq PCR Protocol </p></div></td> | + | <center><td width="85%"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="pcr"></a>Taq PCR Protocol </p></div></td> |
</tr> | </tr> | ||
</table> | </table> |
Revision as of 02:51, 2 October 2012
![](https://static.igem.org/mediawiki/2012/f/fc/UCalgary2012_MainHeaderImage.png)
![](https://static.igem.org/mediawiki/2012/c/c5/UCalgary2012_HeaderLogoOrange.png)
Hello! iGEM Calgary's wiki functions best with Javascript enabled, especially for mobile devices. We recommend that you enable Javascript on your device for the best wiki-viewing experience. Thanks!
Taq PCR Protocol
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.