Ligations done right

John Cumbers recently asked the BioBricks list about how best to run a ligation reaction. Based on the literature and the experience in my lab, here is what we have found works well –

…stick with the normal NEB ligase buffer. Make sure it still has DTT and ATP (not too many freeze/thaw cycles of the BUFFER). Note that the NEB ligase is provided in vast overabundance, and that very little ligase is required. The units tell the story — read them. See this paper for optimization:

Yoshino Y, Ishida M, Horii A. A new 10-min ligation method using a modified buffer system with a very low amount of T4 DNA ligase: the “Coffee Break Ligation” technique. Biotechnol Lett. 2007 Oct;29(10):1557-60. Epub 2007 Jun 21. PMID: 1758103

Note that buffers containing PEG react poorly to heat inactivation of the ligase, inhibiting transformation efficiency.

Posted By: tk

  1. The NEB ligase & ligase buffer are great but before you use the thawed buffer, be sure there is no undissolved precipitate in the tube. If you do see little white bits in the tube, warm it up and keep inverting it until its all dissolved.

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