transforming with RE digest

biocukm at okway.okstate.edu biocukm at okway.okstate.edu
Thu Jul 29 13:17:45 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!!)
>

Several commercial protocols for mutagenesis use restriction digestion of the
total products of the first round of transformation (an oligo mutating the
restriction site had been used in mutagenesis).  The digestion linearizes
non-mutagenized DNAs.  The subsequent transformation is done after a simple
heat inactivation step.  There is no extraction or precipitation.  Works in our
hands.

Ulrich Melcher
Biochem & Mol Biol
Okla. St. Univ.
Stillwater OK USA



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