Ligation inhibiting electroporation?
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Tue Sep 28 11:43:45 EST 1999
In article <firstname.lastname@example.org>, Chris Small
<chris at GENOMEX.COM> writes
>I am ligating high MW DNA (50-200 kb) isolated from a LMP agarose
>pulse field gel and treated with Gelase to a BAC vector (20-50 ng
>insert plus 20-50 ng dephosphorylated vector, 1-2 Units ligase, etc).
>Simple it seems, but even after drop dialysis, my controls suggest
>the ligase (or something in the buffer) is inhibiting
>electroporation. Linearized vector without ligase (testing for uncut
>molecules) yields background blue colonies but when I add ligase, the
>background disappears. If anything, background should increase.
>Ligations look good on a gel so I know recombinants are being
>produced but I don't get the expected number of background blues much
>less whites. All other controls are OK (cells are competent, etc)
This is a known problem with electroporation. Ligase inhibits. Simple
cure. Take one ligation mix add 2x volume of water and heat at 70C for
10 mins to kill ligase. Now zap or EtOH ppt (trace amount of blue
dextran is a nice visible carrier for the pellet), resuspend in water at
required concn. and zap. There have been a variety of papers over the
years about T4 DNA ligase and electroporations in NAR and Biotechniques.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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