Restriction Enzyme Activity and Ligation Problems

Emad Gharavi Emad_G at MSN.COM
Wed May 21 23:05:29 EST 1997

Dear Scott:

One question.  What enzyme are you using for your PCR? I ask because enzymes 
that have 3'-5' exo activity (such as Pfu and Vent) will chew the 3' end of 
your product after the PCR reaction.  During the final 7 min extension at 72 
deg C, terminal residues will be removed leaving a 5' overhang and possibly 
destroying your restriction site.  If you see a band around 350 bp and a 
smear, then this is probably what's happening. This occurs especially if you 
use too much enzyme or when the PCR is run overnight and "soaked" at 4 deg C.  
If this is the case then try self ligating your insert after you've digested 
with Eag1. On a gel you will see a ladder (regardless of whether the digestion 
worked). After self-ligation, clean up the DNA and digest again with Eag I to 
see if you get a single band.  If no Eag I cohesive Eag I sites were generated 
after initial digestion then folowing the second digestion you will still see 
a ladder on a gel.  In this case either the Eag I site is too close to the end 
of your PCR product or the polymerase has destroyed the site. With any 
restriction enzyme I would suggest that you use Taq polymerase for the PCR and 
if that doesn't work add 5-7 to the primers upstream of the digestion site.

Hope that helps.

Emad Gharavi
LSU Medical Center

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