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Team:UEA-JIC Norwich/vectorcloning
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Revision as of 15:32, 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) | pGEM T-easy vector | PCR product | Control Insert DNA | T4 DNA Ligase | Nuclease free water to make final volume of: |
| 5 | 1 | X | - | 1 | 10 |
| 5 | 1 | - | 2 | 1 | 10 |
| 5 | 1 | - | - | 1 | 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
