PCR-vector-sequencing

Stuart Brown browns at ccu.umanitoba.ca
Thu Dec 10 15:28:49 EST 1992


In article <01GRU5I78EQQ8WW4RP at mis.mcw.edu> DMEIER at mis.mcw.edu writes:
>Hello to all,
>  I am designing a set of primers which will give me a PCR product of
>approx. 600 bases.  These primers will have EcoR I and Hind III 
>restriction sites (one only for each primer of course).
>  Is there a plasmid of choice I should put the PCR product in for 
>sequencing?
>  All replies will be gratefully received.
>
>Daniel A. Meier
>DMEIER at mis.mcw.edu

I believe that it is much better to directly sequence your PCR product
using one of the cycle sequencing kits (NEB and BRL both work well) rather
than clone and then sequence.  There has been a fair bit of discussion 
about mutations due to the low fidelity of the Taq polymerase.  If you
clone and then sequence, you have a fair chance of getting a mutated copy
of your gene.  If you sequence directly, then you are sequencing many
thousands (millions?) of different PCR amplified fragments, and the errors
will be averaged out.

If you must clone, then be sure to put your restricion sites far enough 
away from the free ends of your primer.  A lot of enzymes will not cut
a site only 2-4 bases from a free end.

		-Stu

-- 
Stuart M. Brown                             If you can remain cool when all 
U. of Manitoba, Dept. Plant Science         Around you are in panic,
Winnipeg, Manitoba, CANADA
browns at ccu.umanitoba.ca            Then you surely misunderstand the situation



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