cloning PCR products

Sergio seraln at _NO_SPAM_netscape.net
Tue Jan 29 15:16:42 EST 2002



Purvi Bhatt wrote:

> Hi all,
> 
> i too am facing a lot of problems with my 2kb insert which i am trying to
> clone into expression vector. I fail to understand why i get a  lot of
> colonies on vector self ligated plate while the test plate shows less
> colonies. also i am not getting even a single recombinant???? pl help as to
> where  can i be going wrong, is my vector not completely digested or what????
> troubleshooting needed. thanx.
> 
> 
> <http://www.biowww.net/forum/read.php?f=1&i=5283&t=224>
> 

We use to clone the PCR products in a non-expression vector first, with 
blue/white selection. Once we get the correct insert, with no mutations, then we 
clone it into the expression vector. We have observed that when we tried to 
clone it directly in non very tightly represed expression vectors, we always get 
mutant products or religated plasmids, specially with some of our proteins.

Sergio




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