ligation problems (w cut PCR product)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Fri May 26 14:18:13 EST 1995


Sam Hodge writes:
>     Our lab has been having difficuties ligating PCR products 
into vectors. ... It is difficult to know if the PCR 
product has been fully digested as only a few nucleotides have been 
removed from the PCR product....

1. If it won't cut, it's because the extension behind the R. site
isn't long enough.  Try 6 bases.  Don't make them all Gs and Cs
or else you may stabilize a lot of nonspecific priming.

2. The flip side of this problem is that people worry so much about
not being able to cut that they use a huge excess of R. enzyme such
that the trace contaminating activities in it damage the ends and make
them non-ligatable.  Remember that your fold excess is units x hrs/ug. 
(ie. 100 ng cut with 2 ul (10U/ul) for 4 hours is a 800 fold excess) 
You're almost never safe over 100x; and I've seen some spec. sheets
that indicate you're not safe over 5x. If the extension is long
enough, the site will cut just like in any other DNA, so these big
excesses are unnecessary.

You can assay for ligatable ends by doing a self ligation and running
a gel.  You can assay for problem 2 by cutting some control DNA under
the same  conditions, and then trying to religate and run a gel.  

Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San
Antonio
Hardies at uthscsa.edu




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