Help! PCR products cloning

Howard Crawford howard.crawford at mcmail.vanderbilt.edu
Fri Sep 6 10:41:03 EST 1996


My first year in graduate school I had the same problem, so here's a 
technique I invented that works 100% of the time:

Take your PCR products, isolate your fragment on a gel (I prefer 
acrylamide).  Kinase your products and concatamerize them by doing a 
simple ligation.  Now digest with your enzymes and clone. The idea was to 
internalize the sites by more than just a few nucleotides to increase the 
efficiency of the digestion and it really works VERY well.

Of course, my mentor at the time didn't want to publish the technique and 
three years later came by to show me a Biotechniques paper on cloning PCR 
products.  While he was explaining it to me, he says "Oh!  Isn't this 
*your* technique?"  Well, yes, it was.  Anyway the paper worked out the 
math and showed that this technique increases PCR cloning efficiency by 
over 1000-fold.  Give it a shot.

Howard 




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