rapid ligation &electroporation

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Fri Mar 23 15:08:36 EST 2001


thomas.nilsson at kau.se (Thomas Nilsson) wrote:
:Hello,
:
:I am about to do a ligation using the Roche (previously Boehringer) rapid
:ligation kit , followed by electroporation of the ligation product. Roches
:manual states that the ligation reaction has to be purified with the "High
:Pure PCR Product purifiaction kit". Why is this? 

Because they want you to buy their purification kit, that's all.

:A component in the
:ligation kit interfering with the electroporation? 

Rapid ligation buffers are basically the same buffers but
with PEG. Other than salt, there is nothing in ligation reaction
interfering with EP (assuming you inactivate ligase by heating;
PEG at these concentrations does not affect EP). 1 ul of ligation
mixture does not bring up salt high enough to have arcing 
(with properly made EP-competent cells anyway), so it can be 
used directly.

:Anybody with experience
:of electroporating rapid ligations?

We do it here occasionally. If many clones are needed or amounts
of DNA in ligation are pathetically small, then, after 20-30 min 
of "drop dialysis" on 50 micron filters, one can use 10 ul of the 
mix in electroporation.

        - Dima




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