transforming with RE digest

Darren Natale camdna at ubvms.cc.buffalo.edu
Wed Jul 28 10:15:00 EST 1993


In article <1993Jul28.134449.1 at hkucc.hku.hk>, lhyatt at hkucc.hku.hk writes...
>Hi,
>	Can anyone tell me if it is OK to transform competent coli cells
>with a restriction digested sample. I have been reading the recent
>correspondence about checking CIPped vector, and someone suggested digesting
>the ligation product with an enzyme which will only cut the vector without
>insert. I want to remove a restiction site by blunt-ending with Klenow, and in
>the past I have had to screen mostly native vector colonies, and this would
>remove anything which still cut with the enzyme. Do I need to heat
>inactivate, or phenol extract/ ethanol ppt, the ligation/ restiction product
>before transformation. (I did have the required vector, but a bag of samples
>has disappeared during a freezer-defrost, and I can't face screening hundreds
>of minipreps which cut AGAIN!!)

I don't know if you NEED to heat inactivate or phenol extract, but I usually
do (heat inactivate, that is). However, looking in my notes, I realize that
on some occasions I tried to heat inactivate an enzyme that the NEB catalog
says cannot be completely inactivated by this method. I always heat
inactivate the ligase of course :) and adjust buffer conditions to suit the
RE, when feasible (otherwise I do a precipitation before the RE digest).

D. Natale
who will be at camdna at ubvms.cc.buffalo.edu only 2 more days before net
access is lost :(



More information about the Methods mailing list