Question

Tom Knight via methods%40net.bio.net (by tk from mit.edu)
Thu May 8 12:22:47 EST 2008


"juan pablo morera rojas" <morera98 from gmail.com> writes:
> I wanted to ask you about one primer Im working
> with and if you could help me. It has 52bp but I dont know why is so long, I
> found another paper that includes a shorter primer but still I have
> the longer one (it works by the way) besides it includes a enzyme
> restriction site, just adding an "A" in the end of the primer, but
> originally in the sequence there isnt site for any enzyme. If you have some
> clue about it I would really appreciate it.

It is quite common to add additional bases at the 5' end of a primer
which do not match the DNA sequence being amplified.  These extra
bases are incorporated into the PCR product when the complementary
strand is copied.  Likely the last 18-25 bp of your primer will match
the region of template DNA you are amplifying.  That is all that is
required for amplification.  The remaining bases will simply be tagged
onto the product at each end, which allows you to design cloning sites
or other structure into the ends of the PCR product -- a very
powerful, general technique.



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