Inhibition of transformation by ligase - is it true? (Feb/22/2007 )

G'day all,

Does anyone have any evidence for this phenomenon? I was doing some vector-only controls the other day and realised I was putting 10 x vector in my ligase negative transformation compared to my ligase positive transformation. Because I was aliqouting directly from my vector prep compared to aliquoting from my ligation reaction (10 x diluted vector) I was getting many more colonies on my ligase negative control plate. I know some people say ligase inhibits transformation but is this same mistake the real reason why people wrongly say that ligase inhibits transformation or is there actually evidence for this?

Cheers, Rob

at least for electroporation, it is not so much the ligase that inhibits transformation but the PEG in the quick ligase buffer that does the deed. Quick estimates by my labmates and I indicates the PEG in quick ligase causes a 50 fold inhibition of transformation.

Thus in the matter of quick ligase it is a trade off between speed and efficiency. Though quick ligase does have its usefulness particularly with blunt end cloning.

If one electroporates ligation which contains PEG, this migh not work. but if one were to try chemical tranformation, this should not be a problem. There are purification columns to clean up the ligation from PEG for electroporation.

after pelleting DNA with butanol, and as PEG is soluble in ethanol too, it can be efficiently removed by 2washes in 70%EtOH