T/A cloning

Simon TWigger simont at post.its.mcw.edu
Thu Jan 18 15:46:25 EST 1996


>Helkor at facstaff.wisc.edu (Helen Korsmo) says:
>>
>>We ligated an insert into a T-vector (after treating the insert for 30
minutes with Taq; in order to 
>>put A's on the ends) and transformed DH5-alpha cells and plated out on
LB plate with IPTG and 
>>X-gal.  We got several white colonies ( presumably these have insert in
the vector), but mostly 
>>blue (vector alone).  Is it possible for T-vectors to ligate?

It is also possible that the blue colonies have the insert in as well,
but for whatever reason, this hasn't inactivated the B-gal gene. I have
found this on a number of occasions using pGEM-T. If you screen the
whites and some of the blues using colony PCR you find that the insert
is in both. It doesn't always happen and I pick whites in preference to
blues, but if all else fails, check out a few blues and you might be in
luck.

Also you may find that you have light blue colonies (mainly white with
a light blue center for example) as opposed to completely white
ones...quite often these will also have the insert.

Simon.

Simon Twigger, 
Medical College of Wisconsin, Milwaukee, WI.
simont at post.its.mcw.edu



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