LOW EFFICIENCY OF PCR CLONING AND FASLE WHITE COLONIES

a.dodd at auckland.ac.nz a.dodd at auckland.ac.nz
Wed Mar 29 22:03:09 EST 1995


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

What type of enzyme are you using in your PCR? remember not all
thermostable polymerases add A overhangs on PCR products.

I have found that TA cloning kits are a very inefficient and expensive way
of cloning PCR products, I clone my PCR products using any blunt cut
vector that is available pUC18, pGEM etc. I blunt end my PCR fragments
using a quick 30 min incubation with pfu polymerase (better than klenow
for blunting) and T4 kinase before ligation. this works well and manage to
get greater than 25% white colonies. This procedure is good because it
costs very little in comparison to a TA cloning kit and works better.

I hope this is some help

Andrew Dodd
a.dodd at auckland.ac.nz



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