Problems of XbaI-KpnI cloning into pGEM-3, pBSK(-)
Michael J. Tuvin
mtuvin at mbcr.bcm.tmc.edu
Fri Dec 17 12:56:09 EST 1993
Dear folks! I cannot get any colonies ...
My fragments (1,6 to 3,5 kb) were PCR-made, cleaned with Centricons, digested
with XbaI+KpnI (I saw the end-pieces of 33 bp and 64 bp on a NuSieve gel, so I
am really sure both enzymes cut), purified out of the SeaKem agarose gel with
Qiagen-kit. Vectors were cut with the same enzymes and also purified out of the
agarose. I took 400 ng of vector and 1500 ng of insert per 5 ul ligation reaction,
0.5 ul T4 DNA Ligase (NEB), 16 degrees o/n. Gel shows disappearence of the vector
band and the whole ladder of bands of higher mol. weight.
Transformations were done on competent DH5-alpha's and TG-1's with 1ul of 1:5
dilution. Appropriate controls look just as they should ...
What could be my problem?! Begging for any clues.
Please e-mail responses to mtuvin at mbcr.bcm.tmc.edu (-those I could read even at home!!)
or post on the newsgroup.
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