MY PCR product
Dr. Duncan Clark
duncan at genesys.demon.co.uk
Sat Aug 9 05:06:02 EST 1997
In article <33EB4C63.4412 at shef.ac.uk>, A Al-dukhyil <MBQ96AA at shef.ac.uk>
>I wonder if any one could find a suitable solution for a PCR product of
>2Kb gene I designed a SmaI site before 5'end of this PCR product and
>EcoRI before 3'end of it. Then I ligated this blunt end PCR
>product(because of using Vent Polymerase) in PBR322 plasmid at EcoRV site
>but when I tried to recover my PCR product by digestion with SmaI then
>EcoRI it wasn't cutted with SmaI!
Assuming the oligo is correct then the most likely problem is that the
Vent proof-reading exo is having a nibble at your SmaI oligo. Try
repeating the PCR but add the Vent at around 90C in your PCR. It keeps
the exo from having a go in the time it takes you to set up the
reactions, load the PCR machine and to get beyond say 75C.
This exo digestion of oligos is a known problem when using Vent or Pfu
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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