PCR product (SmaI) site
M.Champagne at nospam.cellbio.duke.edu
Tue Aug 19 06:01:37 EST 1997
In article <33ECECFA.4296 at shef.ac.uk>, A Al-dukhyil <MBQ96AA at shef.ac.uk> wrote:
> Dear everybody,
> I wonder if any one could help me in this problem:
> I did PCR by using Vent Pol. for a gene of 2Kb with insertion of SmaI
> site near 5'end. I got good PCR product then I inserted this product in
> PBR322 plasmid (EcoRV) site. I had the right size 6.3Kb BUT,when I cutted
> with SmaI I got uncut plasmid. I hope to find good solution for this
> Note: SmaI enz. has been tested and it was alright.
> Thanks for helping
I'm not sure i got your story right, but if you expected a Sma I fragment
ligated into an EcoRV fragment (both blunt) to be recleaved, there's your
problem! Sma I cuts CCC/GGG, Eco RV cuts GAT/ATC. They will ligate since
the products are blunt, but not recleave with either enzyme since the new
product will be CCCATC, which is not recognize by Sma I or Eco RV.
Mon pays ce n'est pas un pays, c'est l'hiver (Vigneault).
Pls remove -nospam- from my address if you reply by e-mail
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