LOW EFFICIENCY OF PCR CLONING AND FASLE WHITE COLONIES

[John M. Freeman]

In article <Pine.OSF.3.91a.950328112741.29264A-100000 at christa.unh.edu>,
xz at CHRISTA.UNH.EDU (Xin-Hua Zhou) wrote:

> Hi, Netter:
> I am using Invitrogen TA cloning kit to clone my PCR products (400-500bp) 
> with very low efficency (2%). PCR and restriction analysis did not 
> show expected bands when I pick up white colonies to isolate plasmids. I 
> would like to have your suggestions on this problem.
>  Thank you in advance!
> 
>   Zhou, UNH

I have been using the TA cloning kit and cloning in pieces from 200 to 1300
bp.
I usually get about 25% white clones and then about 20% of those have my
insert.  I use a Quick Check protocal I found in Biotechniques to help me
screen my clones and it works well.  I isolate my PCR product band from the
gel with Gene Clean and then ligate it.  I wish my efficiencies were
higher.  I talked to a researcher at the AACR meeting and he thought that
the short length of the overhang (the T's)  was the main reason for the
poor lign eff.  He used a different plasmid and made cuts giving him about
12 bp of overhang.  He thought this was a better way of doing it.  Good
luck.  Post if you find a better method for working with the TA cloning
kit.

John M. Freeman
UT Memphis
email: jjain at utmem1.utmem.edu