Team:UEA-JIC Norwich/vectorcloning
From 2011.igem.org
(Difference between revisions)
(Created page with "{{Banner}} <html> 1. Centrifuge the pGEM-T easy vector and control insert DNA tubes to collect contents at the bottom of the tubes.<br> 2. Ligation reactions set up as described ...") |
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<tr> | <tr> | ||
<td>Ligase Buffer(vortex first)</td> | <td>Ligase Buffer(vortex first)</td> | ||
- | <td> | + | <td>5</td> |
- | <td> | + | <td>5</td> |
- | <td> | + | <td>5</td> |
- | + | ||
- | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>pGEM-T vector</td> |
<td>1</td> | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PCR Product</td> | ||
<td>X</td> | <td>X</td> | ||
<td>-</td> | <td>-</td> | ||
- | <td> | + | <td>-</td> |
- | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>Control Insert DNA</td> |
- | + | ||
<td>-</td> | <td>-</td> | ||
<td>2</td> | <td>2</td> | ||
- | <td> | + | <td>-</td> |
- | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>T4 DNA Ligase</td> |
<td>1</td> | <td>1</td> | ||
- | |||
- | |||
<td>1</td> | <td>1</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Nuclease free water up to;</td> | ||
+ | <td>10</td> | ||
+ | <td>10</td> | ||
<td>10</td> | <td>10</td> | ||
</tr> | </tr> |
Revision as of 15:38, 21 September 2011
UNIVERSITY OF EAST ANGLIA-JOHN INNES CENTRE
1. Centrifuge the pGEM-T easy vector and control insert DNA tubes to collect contents at the bottom of the tubes.
2. Ligation reactions set up as described below:
Reaction Component (µl) | Standard Reaction (µl) | Positive Control (µl) | Background control (µl) |
---|---|---|---|
Ligase Buffer(vortex first) | 5 | 5 | 5 |
pGEM-T vector | 1 | 1 | 1 |
PCR Product | X | - | - |
Control Insert DNA | - | 2 | - |
T4 DNA Ligase | 1 | 1 | 1 |
Nuclease free water up to; | 10 | 10 | 10 |
3. Calculate the concentration of insert via formula;
(50ng × kb size of insert/kb size of vector) × 3/1 = concentration required
4. From this use the appropriate volume of sample.
5. Incubate at either room temperature for 60 minutes or 16 hours at 4˚C