Team:UEA-JIC Norwich/vectorcloning

From 2011.igem.org

Revision as of 15:38, 21 September 2011 by Awalsham (Talk | contribs)

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