Subcloning problems

ELADER at AMBION.COM ELADER at AMBION.COM
Thu Mar 19 16:21:41 EST 1998


a) your digestion and gel purification of the insert was no good. There is no
other way to explain how you could recover the cloning plasmid from a sceen
for the expression plasmid.

b) Your double digestion of the expression plasmid prior to cloning was also
no good because the expression clones you recovered had no insert. A properly
double digested plasmid should have very low background.

c) The probe you used to do a colony screen with was contaminated with vector
since you picke up positive clones that did not have insert.

Solutions?

a) make sure double digestions are actually double digestions. Cut a pilot
digestion of each plasmid under the double digestion conditions but using each
enzyme seperately. Each should cut to completion. Only then can you be
confident that the double cut is actually cutting both sites.

b) Think hard about why your gel purification of the insert didnt work. Is it
close in size to the vector? If you can't get a clen seperation, consider
cutting the reaction mix with another enzyme (in addition to the 2 which
releases the insert) which does not cut the insert but cuts the vector into
smaller chunks. Your gel purification should then be easier and cleaner.

c) make sure you run appropriate controls. You had some weird results for
sure. Do a transformation with insert alone, insert ligated, vector alone,
vector ligated, vector plus insert ligated. This way you can assess whether
yuo have uncut plasmid anywhere, whether you have single cut plasmid
religating.

d) colony screening is not a bad idea. however, maybe add a control plate of
colonies containing insert-less plasmids of both types. This strikes me as
overkill. However, agian, your data seemed quite bizarre.

Eric

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