Ligations - help

Edgar Valencia 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.
> Help!
> 
> 
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
CIFN-UNAM
Mexico



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