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Team:Groningen/protocols/PCR
From 2013.igem.org
(Difference between revisions)
| Line 41: | Line 41: | ||
<td>Final concentration</td> | <td>Final concentration</td> | ||
</tr> | </tr> | ||
| - | <tr><td> | + | <tr><td>MQ water</td> |
<td>up to 50μl</td> | <td>up to 50μl</td> | ||
<td></td> | <td></td> | ||
| Line 54: | Line 54: | ||
</tr> | </tr> | ||
<tr><td>F-primer</td> | <tr><td>F-primer</td> | ||
| - | <td> | + | <td>2.5μl</td> |
<td>0.2μM</td> | <td>0.2μM</td> | ||
</tr> | </tr> | ||
<tr><td>R-primer</td> | <tr><td>R-primer</td> | ||
| - | <td> | + | <td>2.5μl</td> |
<td>0.2μM</td> | <td>0.2μM</td> | ||
</tr> | </tr> | ||
| Line 66: | Line 66: | ||
</tr> | </tr> | ||
<tr><td>Phusion Pol. 2U/μl</td> | <tr><td>Phusion Pol. 2U/μl</td> | ||
| - | <td>0.5μl</td> | + | <td>0.3μl</td> |
| + | <td>0.02U/μl</td> | ||
| + | </tr> | ||
| + | <tr><td>DMSO 5#</td> | ||
| + | <td>1.5μl</td> | ||
<td>0.02U/μl</td> | <td>0.02U/μl</td> | ||
</tr> | </tr> | ||
Revision as of 10:52, 27 July 2013
PCR
Materials:
- PCR tubes
- F- and R-primer
- DNTPs
- Phusion buffer
- MQ water
- Phusion Polymerase
- DNA template
Steps:
| Component | 50μl reaction | Final concentration |
| MQ water | up to 50μl | |
| 5x Phusion Buffer | 10μl | 1x |
| 10mM dNTPs | 1μl | 200μM |
| F-primer | 2.5μl | 0.2μM |
| R-primer | 2.5μl | 0.2μM |
| Template DNA | 1μl | ~1ng |
| Phusion Pol. 2U/μl | 0.3μl | 0.02U/μl |
| DMSO 5# | 1.5μl | 0.02U/μl |
Cycling instructions:
| Temperature | Time | Number of cycles |
| 98°C | 30sec | 1 |
| 98°C | 10sec | 30 |
| primer annealing temperature | 30sec | |
| 72°C | 30sec/kb | |
| 72°C | 10min | 1 |
| 4°C | ∞ | 1 |
Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/f-530s-product-information.pdf
iGEM 2013 Groningen
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