DNA Insert Ligation into Vector DNA (with T4 DNA Ligase)
- In a microcentrifuge tube prepare 5-10µl mix in water or TE buffer of digested (if preferred dephosphorylated and purified) vector DNA (50-400ng) and foreign DNA to be inserted.
- Add the following components to the same tube:
- Incubate the mixture for 1 hour at 22°C.
- Inactivate T4 DNA Ligase by heating reaction mixture at 65°C for 10 minutes.
- Use the mixture for transformation.
- TE buffer: 10mM Tris-HCl, 1mM EDTA (pH 7.8).
- Use equal or higher (up to 3-fold) molar concentration of insert DNA termini over vector DNA.
- If the yield of ligation product is insufficient, prolong the reaction time (overnight).
- The resulting ligation reaction mixture can be used directly for bacterial transformation. An excess of ligation mixture in regard to competent cells may decrease the transformation efficiency. It is necessary to extract DNA with chloroform and precipitate with ethanol prior to electro-transformation.
- Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
- Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 1994-2001.
Updated August 23, 2002 13:51