LOW EFFICIENCY OF PCR CLONING AND FASLE WHITE COLONIES

Malcolm Campbell malcolm at unity.ncsu.edu
Tue Mar 28 17:34:21 EST 1995


In article <Pine.OSF.3.91a.950328112741.29264A-100000 at christa.unh.edu>,
xz at CHRISTA.UNH.EDU (Xin-Hua Zhou) writes:
Subject: LOW EFFICIENCY OF PCR CLONING AND FASLE WHITE COLONIES

>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.

We have had similar experiences here for some primer pairs.  Our
hypothesis is that we are getting small (ie. <50 bp) PCR products which
we don't see on gels designed to look at larger products.  Nonetheless,
we hypothesize that these smaller products are in molar excess of the
predicted products and therefore effectively compete with the desired
products for ligation into the TA cloning vector.  The small fragments
are therefore cloned, give white colonies but do not give the predicted
restriction pattern or PCR products from the plasmids in the colonies. 
Gel purifying the product of the predicted size should clear up this
problem.  

Best of luck,

Malcolm

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