Ligation inhibiting electroporation?
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Thu Sep 30 03:01:39 EST 1999
In article <lmarek-2909991000110001 at jacob.agron.iastate.edu>, Laura F.
Marek <lmarek at iastate.edu> writes
>I have made a couple of BAC libraries now and Duncan Clark's suggestion of
>heat killing the ligase is a very good one. I never try to precipitate
>the ligated BAC DNA - too big or whatever - just heat kill for 10 min at
>about 65C. Sometimes I do a drop dialysis of the ligation after the heat
>kill before electroporating; sometimes I just dilute the ligation with
>some water (empirically determine the appropriate dilution, usually around
>1 in 5) and then electroporate.
The dilution with water we do is solely to drop the conductivity of the
ligation mix so the electroporator won't arc across the gap. We normally
EtOH ppt with blue dextran carrier as we use plasmids not large BACs.
I've never tried the butanol extraction suggested in this thread but
have seen it in the literature so I would try either method and take the
one that works best for you.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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