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Ultrasonic Ligation

Marianna Max drmax at casbah.acns.nwu.edu
Wed Aug 11 20:08:47 EST 1993


There seems to be lots of confusion out there about ligation protocols.
Wildtype T4 ligase had inherent endonuclease activity and ligations were
incubated at 16oC to block the endonuclease activity. This also slowed to
ligation reaction since the ligase likes something closer to roomtemp for
maximal activity. The T4 ligase we use today is from a recombinant which no
longer has the endonuclease activity. There is no reason to incubate at
16oC anymore (unless for some reason you want to slow down the ligation
reaction!). 

If you are very very concerned to get every last bit of your DNA ligated
then maybe you might want to resort to longer incubations or ultrasonic
cleaners but for run-of-the-mill stuff where you just need to get a
particular fragment subcloned or a PCR product into a vector, a few minutes
on the bench is sufficient.

Max



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