Restriction Enzyme Activity and Ligation Problems

Darren Natale dnatale at
Thu May 22 07:50:03 EST 1997

Emad Gharavi wrote:

> 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).

This is only true if the primers are phosphorylated, or if the fragment
phosphorylated after PCR.  Otherwise, only digested fragments will


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