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Team:Groningen/protocols/PCR
From 2013.igem.org
(Difference between revisions)
| Line 48: | Line 48: | ||
<tr> | <tr> | ||
<td>MQ water</td> | <td>MQ water</td> | ||
| - | <td | + | <td>up to 50 µl</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 54: | Line 54: | ||
<tr> | <tr> | ||
<td>5x Phusion Buffer</td> | <td>5x Phusion Buffer</td> | ||
| - | <td | + | <td>10 µl</td> |
<td>1x</td> | <td>1x</td> | ||
</tr> | </tr> | ||
| Line 60: | Line 60: | ||
<tr> | <tr> | ||
<td>DMSO 5%</td> | <td>DMSO 5%</td> | ||
| - | <td | + | <td>1.5 µl</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 66: | Line 66: | ||
<tr> | <tr> | ||
<td>10 mM dNTPs</td> | <td>10 mM dNTPs</td> | ||
| - | <td | + | <td>1µl</td> |
<td>200 µM</td> | <td>200 µM</td> | ||
</tr> | </tr> | ||
| Line 72: | Line 72: | ||
<tr> | <tr> | ||
<td>primer F</td> | <td>primer F</td> | ||
| - | <td | + | <td>2.5 µl</td> |
<td>0.5 µM</td> | <td>0.5 µM</td> | ||
</tr> | </tr> | ||
| Line 78: | Line 78: | ||
<tr> | <tr> | ||
<td>primer R</td> | <td>primer R</td> | ||
| - | <td | + | <td>2.5 µl</td> |
<td>0.5 µM</td> | <td>0.5 µM</td> | ||
</tr> | </tr> | ||
| Line 84: | Line 84: | ||
<tr> | <tr> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
| - | <td | + | <td>1 µl</td> |
<td>∼1 ng</td> | <td>∼1 ng</td> | ||
</tr> | </tr> | ||
| Line 90: | Line 90: | ||
<tr> | <tr> | ||
<td>Phusion Polymerase <br>2U/µl</td> | <td>Phusion Polymerase <br>2U/µl</td> | ||
| - | <td | + | <td>0.3 µl</td> |
<td>0.01U/µl</td> | <td>0.01U/µl</td> | ||
</tr> | </tr> | ||
Revision as of 12:10, 9 September 2013
PCR
Materials:
- PCR tubes
- MQ water
- DMSO 5%
- Phusion buffer
- DNTPs
- F- and R-primer
- Phusion Polymerase
- DNA template
PCR reaction mixture:
| Component | 50 µl | Final concentration |
|---|---|---|
| MQ water | up to 50 µl | |
| 5x Phusion Buffer | 10 µl | 1x |
| DMSO 5% | 1.5 µl | |
| 10 mM dNTPs | 1µl | 200 µM |
| primer F | 2.5 µl | 0.5 µM |
| primer R | 2.5 µl | 0.5 µM |
| template DNA | 1 µl | ∼1 ng |
| Phusion Polymerase 2U/µl |
0.3 µl | 0.01U/µl |
Cycling instructions:
| Temperature | Time | Number of Cycles |
|---|---|---|
| 98°C | 2 min | 1 |
| 98°C | 10 sec | 34 |
| Tm-5°C | 25 sec | |
| 72°C | 30 sec/kb | |
| 72°C | 10 min | 1 |
| 4°C | ∞ | 1 |
Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/f-530s-product-information.pdf
iGEM 2013 Groningen
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