Team:UEA-JIC Norwich/vectorcloning

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4. From this use the appropriate volume of sample.<br>
4. From this use the appropriate volume of sample.<br>
5. Incubate at either room temperature for 60 minutes or 16 hours at 4˚C<br>
5. Incubate at either room temperature for 60 minutes or 16 hours at 4˚C<br>
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http://www.promega.com/~/media/Files/Resources/ProtCards/pGEM-T%20and%20pGEM-T%20Easy%20Vector%20Systems%20Quick%20Protocol.ashx
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Latest revision as of 21:26, 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


http://www.promega.com/~/media/Files/Resources/ProtCards/pGEM-T%20and%20pGEM-T%20Easy%20Vector%20Systems%20Quick%20Protocol.ashx