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Team:Groningen/protocols/Colony PCR
From 2013.igem.org
(Difference between revisions)
| Line 42: | Line 42: | ||
<tr> | <tr> | ||
<th>Component</th> | <th>Component</th> | ||
| - | <th>20µl</th> | + | <th>20 µl</th> |
<th>Final concentration</th> | <th>Final concentration</th> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td>MQ water</td> | <td>MQ water</td> | ||
| - | <td ALIGN=CENTER>up to 20µl</td> | + | <td ALIGN=CENTER>up to 20 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 ALIGN=CENTER>2µl</td> | + | <td ALIGN=CENTER>2 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 ALIGN=CENTER>1.5µl</td> | + | <td ALIGN=CENTER>1.5 µl</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>10 mM dNTPs</td> |
| - | <td ALIGN=CENTER>1µl</td> | + | <td ALIGN=CENTER>1 µl</td> |
<td>200µM</td> | <td>200µM</td> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td>primer F</td> | <td>primer F</td> | ||
| - | <td ALIGN=CENTER>1µl</td> | + | <td ALIGN=CENTER>1 µl</td> |
| - | <td>0.5µM</td> | + | <td>0.5 µM</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>primer R</td> | <td>primer R</td> | ||
| - | <td ALIGN=CENTER>1µl</td> | + | <td ALIGN=CENTER>1 µl</td> |
| - | <td>0.5µM</td> | + | <td>0.5 µM</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
| - | <td ALIGN=CENTER>1µl</td> | + | <td ALIGN=CENTER>1 µl</td> |
| - | <td>∼ | + | <td>∼1 ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Phusion Polymerase <br>2U/µl</td> | <td>Phusion Polymerase <br>2U/µl</td> | ||
| - | <td ALIGN=CENTER>0.3µl</td> | + | <td ALIGN=CENTER>0.3 µl</td> |
<td>0.01U/µl</td> | <td>0.01U/µl</td> | ||
</tr> | </tr> | ||
| Line 108: | Line 108: | ||
<tr> | <tr> | ||
<td>98°C</td> | <td>98°C</td> | ||
| - | <td ALIGN=CENTER> | + | <td ALIGN=CENTER>10 min</td> |
<td>1</td> | <td>1</td> | ||
</tr> | </tr> | ||
| Line 114: | Line 114: | ||
<tr> | <tr> | ||
<td>98°C</td> | <td>98°C</td> | ||
| - | <td ALIGN=CENTER> | + | <td ALIGN=CENTER>10 sec</td> |
<td rowspan="3">34</td> | <td rowspan="3">34</td> | ||
</tr> | </tr> | ||
| Line 120: | Line 120: | ||
<tr> | <tr> | ||
<td>T<sub>m</sub>-5°C</td> | <td>T<sub>m</sub>-5°C</td> | ||
| - | <td ALIGN=CENTER> | + | <td ALIGN=CENTER>25 sec</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>72°C</td> | <td>72°C</td> | ||
| - | <td ALIGN=CENTER> | + | <td ALIGN=CENTER>30 sec/kb</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>72°C</td> | <td>72°C</td> | ||
| - | <td ALIGN=CENTER> | + | <td ALIGN=CENTER>10 min</td> |
<td>1</td> | <td>1</td> | ||
</tr> | </tr> | ||
Revision as of 15:00, 27 September 2013
Colony PCR
Materials:
- PCR tubes
- MQ water
- DMSO 5%
- Phusion buffer
- DNTPs
- F- and R-primer
- Phusion Polymerase
- DNA template
PCR reaction mixture:
| Component | 20 µl | Final concentration |
|---|---|---|
| MQ water | up to 20 µl | |
| 5x Phusion Buffer | 2 µl | 1x |
| DMSO 5% | 1.5 µl | |
| 10 mM dNTPs | 1 µl | 200µM |
| primer F | 1 µl | 0.5 µM |
| primer R | 1 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 | 10 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 |
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