what's the best way to clone PCR products?

Tracy Aquilla aquilla at salus.med.uvm.edu
Mon Sep 26 17:59:20 EST 1994


In Article <80705041wnr at genesys.demon.co.uk>, Duncan at genesys.demon.co.uk
(Duncan Clark) wrote:
>It's all very well saying add 5-8bp per oligo but by the time you have 
>done this for say 10 PCR products then, depending on your facility, the
>extra oligo costs could have paid for a TA kit and you don't have to 
>clean up the PCR product either.
>
>Duncan 
>
>-----------------------------------------------------------------------------
>Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
>G4ELJ                               | Compuserve:  100015.1406 at compuserve.com
>-----------------------------------------------------------------------------
>

That's why I generally don't add ANY extra bases to my oligos. Usually, I
can create a useful RE site by placing a mismatched base or two somewhere
near the middle of the oligo. If that makes the Tm go down too much, I just
extend a base or two from one of the ends. I have had excellent success
using mutant oligos with RE sites engineered into them this way, and have
thus saved plenty (more than the cost of a kit) on per base charges.
Cleaning up a PCR product is trivial, and only takes about ten minutes.
Subcloning from one vector into another, as many people must do with their
TA-cloning kits, takes at least an extra day, and time is money. We do have
a TA cloning kit sitting in the freezer, but I haven't even touched it.
Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu



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