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Team:Groningen/protocols/PCR
From 2013.igem.org
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<div class="mainContent"> | <div class="mainContent"> | ||
| - | < | + | <h2>PCR</h2> |
<h5>Materials:</h5> | <h5>Materials:</h5> | ||
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<li>DNA template</li> | <li>DNA template</li> | ||
</ul> | </ul> | ||
| + | |||
| + | |||
<p> | <p> | ||
<br> | <br> | ||
<h5>PCR reaction mixture:</h5> | <h5>PCR reaction mixture:</h5> | ||
| - | <table | + | <table id="normal" width="60%"> |
<tr> | <tr> | ||
<th>Component</th> | <th>Component</th> | ||
| - | <th>50 | + | <th>50 ml</th> |
| - | <th>Final concentration</th> | + | <th>Final concentration</th> |
</tr> | </tr> | ||
<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 56: | ||
<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> | ||
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<tr> | <tr> | ||
<td>DMSO 5%</td> | <td>DMSO 5%</td> | ||
| - | <td | + | <td>1.5 µl</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>10 mM dNTPs</td> |
| - | <td | + | <td>1 µl</td> |
| - | <td>200µM</td> | + | <td>200 µM</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>primer F</td> | <td>primer F</td> | ||
| - | <td | + | <td>2.5 µl</td> |
| - | <td>0.5µM</td> | + | <td>0.5 µM</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>primer R</td> | <td>primer R</td> | ||
| - | <td | + | <td>2.5 µl</td> |
| - | <td>0.5µM</td> | + | <td>0.5 µM</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
| - | <td | + | <td>1 µl</td> |
| - | <td>∼ | + | <td>∼1 ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>Phusion Polymerase <br> | + | <td>Phusion Polymerase <br>2 U/µl</td> |
| - | <td | + | <td>0.3 µl</td> |
| - | <td>0. | + | <td>0.01 U/µl</td> |
</tr> | </tr> | ||
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<br> | <br> | ||
<h5>Cycling instructions:</h5> | <h5>Cycling instructions:</h5> | ||
| - | <table | + | <table id="normal" width="60%"> |
<tr> | <tr> | ||
<th>Temperature</th> | <th>Temperature</th> | ||
| Line 108: | Line 110: | ||
<tr> | <tr> | ||
<td>98°C</td> | <td>98°C</td> | ||
| - | <td> | + | <td>2 min</td> |
| - | <td> | + | <td>1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>98°C</td> | <td>98°C</td> | ||
| - | <td> | + | <td>10 sec</td> |
<td rowspan="3">34</td> | <td rowspan="3">34</td> | ||
</tr> | </tr> | ||
| Line 120: | Line 122: | ||
<tr> | <tr> | ||
<td>T<sub>m</sub>-5°C</td> | <td>T<sub>m</sub>-5°C</td> | ||
| - | <td> | + | <td>25 sec</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>72°C</td> | <td>72°C</td> | ||
| - | <td> | + | <td>30 sec/kb</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>72°C</td> | <td>72°C</td> | ||
| - | <td> | + | <td>10 min</td> |
<td>1</td> | <td>1</td> | ||
</tr> | </tr> | ||
Latest revision as of 23:25, 4 October 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 ml | Final concentration |
|---|---|---|
| MQ water | up to 50 µl | |
| 5x Phusion Buffer | 10 µl | 1x |
| DMSO 5% | 1.5 µl | |
| 10 mM dNTPs | 1 µl | 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 2 U/µl |
0.3 µl | 0.01 U/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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