PCR product (SmaI) site

Marc Champagne 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 
> problem.
> 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.

m

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