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ligation inhibitors - (Mar/22/2006 )

I'm having problems with a ligation.
I've heard that the TBE buffer can inhibit the ligation reaction. Can someone confirm this assumption?
the DNA fragment which I'm cloning is a gene fusion with the GFP. Can the GFP (N-terminal fused) be toxic for the E .coli strain?


EDTA will surely inhibits ligation.
ligation needs a transfert of phoshpate. You surely know that cleaage of ATP in ADP + Pi needs magnesium.
EDTA loves divalent cations so feels comfortable with Mg++...


I've had experiences where GFP has poisoned my E.coli when subcloning for other species.

For example, some staph operators do not work like they do in E. coli and you can get a promoter that is super expressed and the GFP poisoned the E. coli cells. But, that occured with full length reporter. However, you're fusion may be toxic in the same way regardless of what gene it is.