Ligations - help
edgar at cifn.unam.mx
Wed Jun 5 14:48:45 EST 1996
Hi Dr. Jane:
On Wed, 5 Jun 1996, Jane McHarg wrote:
> I have been trying to ligate a 3.1kb mutant pcr fragment into a
> homemade yeast/Ecoli shuttle expression vector unsuccessfully for
> weeks. The vector has a Bam H1 site and is 7.5kb. It is useful as it
> promotes my gene under its own promoter and as a result 50 -80% of the
> total cell protein is my gene product, so I must use this vector.
> Other people have successfully used this system. The pcr frag is T7 -
> T3 and I then cut it with Bam H1 - I can confirm it has cut on a gel.
Did you clean your fragment and vector after BamHI digestion? Perhaps the
salts in the buffer are interfering with your ligase.
Did you tried with different ratios of fragment/vector?
Did you check your ligation in a gel, or what you do is transform or
electroporate your cells and you get no transformats? If you check your
ligation in a gel Are you sure your cells are competent?
Finally, with such level of expression are you sure your protein is not
toxic or lethal to E. coli?
I hope this helps
Edgar Valencia Morales
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