Tuesday, November 18, 2003

Description
Appropriate vector DNA is digested with the relevent restriction endonucleases and the 5'-terminal phosphates removed by CIP. The DNA fragment to be cloned is typically prepared by endonuclease digestion or PCR, electrophoretic separation and purification.

Procedure
1) Set up the ligation reaction (typically 10ul final volume) by adding the following to a sterile, eppendorf tube:-

50ng of de-phosphorylated vector DNA (typically 2ul)
2ul 5 x T4 DNA Ligase Buffer
nul insert DNA (sufficient insert DNA should be added to give a 3:1 insert:vector molar ratio)
(5-n)ul sterile, nano-pure H2O
1 unit T4 DNA ligase (typically 1ul)

2) Incubate the ligation overnight at 15C (cohesive terminii may be sufficiently ligated after as little as 1 hour)

Reaction products can either be used directly for transformation of E. coli or frozen at -20C for later use.



Recipes
T4 DNA Ligase (Gibco-BRL)
5 x T4 DNA Ligase Buffer (25mM MgCl2, 100mM Tris.Cl (pH7.7), 50mM DTT, 2.5mM ATP, 25% PEG 8000, Gibco-BRL)
Sterile, nano-pure water



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