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Team:UEA-JIC Norwich/vectorcloning
From 2011.igem.org
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
