pGEM-T cloning

John Troyer nestgrp at earthlink.net
Tue Jul 9 09:22:56 EST 1996


"Miss. V.M. Hodges" <vhodges at hgmp.mrc.ac.uk> wrote:


>I've recently switched from using InVitrogen's TA kit to the pGEM-T 
>vector from Promega to get both the sp6 and T7 promoters in one vector.
>I've found that in my hands the cloning efficiency of the pGEM vector is 
>really low despite having tried a number of vector:insert ratios.
>Has anyone any suggestions?
>The transformation efficiency was good but  very few colonies contained 
>inserts. I have been using blue-white selection but I'm 
>getting very few colonies on my test plates to start off with and most of 
>the colonies were blue . Even clonies that were white had small blue 
>centres but still had no insert.


Vivian:

In my experience, two things are important in optimizing pGEM-T
cloning.  First, I have found that some clean up of the PCR product
greatly increases ligation efficiency with this product.  A simple
clean up to remove unincorporated dNTPs from the PCR product is
sufficient.   Second, make sure your host cells are grown on minimal
media supplemented with thymidine (M9 media plus thymidine) prior to
making them competent to maintain the proper genotype.  If they are
grown on rich media (ie LB) you will allow some of the cells to revert
resulting in white colonies even without an insert in the lacZ gene.

regards,

John




--------------------------------------------------------
 John K. Troyer, Ph.D.    The Nest Group, Inc.        
 Orders: 800-347-6378     Tech Support: (410)625-3782 
          e-mail: nestgrp at earthlink.net               
Visit our WWW Page at  http://world.std.com/~nestgrp  
-------------------------------------------------------




More information about the Methods mailing list