Restriction Enzyme Activity and Ligation Problems
Emad_G at MSN.COM
Wed May 21 23:05:29 EST 1997
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.
LSU Medical Center
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