Team:UEA-JIC Norwich/vectorcloning

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(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>pGEM T-easy vector</td>
+
<td>5</td>
-
<td>PCR product</td>
+
<td>5</td>
-
<td>Control Insert DNA</td>
+
<td>5</td>
-
<td>T4 DNA Ligase</td>
+
-
<td>Nuclease free water to make final volume of:</td>
+
</tr>
</tr>
<tr>
<tr>
-
<td>5</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>1</td>
+
<td>-</td>
-
<td>10</td>
+
</tr>
</tr>
<tr>
<tr>
-
<td>5</td>
+
<td>Control Insert DNA</td>
-
<td>1</td>
+
<td>-</td>
<td>-</td>
<td>2</td>
<td>2</td>
-
<td>1</td>
+
<td>-</td>
-
<td>10</td>
+
</tr>
</tr>
<tr>
<tr>
-
<td>5</td>
+
<td>T4 DNA Ligase</td>
<td>1</td>
<td>1</td>
-
<td>-</td>
 
-
<td>-</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-JIC

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

Retrieved from "http://2011.igem.org/Team:UEA-JIC_Norwich/vectorcloning"