adding non-palindromic r.e site to primer

arasu a_thiruna at hotmail.com
Fri Oct 8 13:58:31 EST 2004


yes, i figured out the same- but it was too late- i had already made
the primers, ligated and tried transformation couple of times and when
i was posting yesterday, i got 3 colonies from one high insert:vector
ratio transformation- out of which two colonies shows expected
restriction profile (thank god!)-i have to pcr to further check- but i
guess it worked somehow and i am assuming the bacteria might have
repaired it.

thanks for confirming my line of thought- bcos it was driving me
little nuts


arasu



Tom Knight <tk at csail.mit.edu> wrote in message news:<vuy655l5rkj.fsf at soggy-fibers.csail.mit.edu>...
> a_thiruna at hotmail.com (arasu) writes:
> 
> > Hi, i dont know whether i am missing something here- if non
> > palindromic  r.e site is added to both forward and reverse primers
> > (both have the same additional nucleotides at the 5' end), in the
> > final ligation will there be a problem- i was trying for enzyme
> > bsu361, when i was writing out the sites and aligning the ligation on
> > paper- one base pair was not aligning-will it be repaired in the
> > bacterial host or am i missing something.
> 
> You can try this, but I think you have a problem.  The cut site for Bsu36I
> is CC'TNAGG.  If you have the same sequence (say, CCTGAGG) on each primer,
> the cut PCR product will have ends that look like this:
> 
> TGAGG----CC
>    CC----GGAGT
> 
> Assume your vector has a site which is also CCTGAGG.
> 
> Your vector ends will be:
> 
> --CC       TGAGG---
> --GGACT       CC---
> 
> The left ligation will work, but the right one will not.
> With only a 3 base overhang, I'd doubt if you can make this work.



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