direct phage sequencing

Stuart Brown browns at ccu.umanitoba.ca
Fri Sep 18 10:57:51 EST 1992


In article <1992Sep16.141612.13559 at gserv1.dl.ac.uk> CALCIUM at FRCICT81.EARN writes:
>hello to everybody,
> 
> 
>I would like to obtain the sequence of a genomic clone in the phage EMBL3 using
>internal oligonucleotides. Could you please send me advice (quality of
>DNA, type of purification, quantity of DNA...). Thank you in advance.
> 
>Jacqueline

I tried direct sequencing of phage DNA by several different methods without 
any luck.  What did work nicely was to amplify a region from the phage by
PCR and then use cyle sequencing on the PCR product (I used NEB's Vent
polymerase kit). Most people reccomend using nested primers -ie. PCR with one
set of primers and then sequence with an internal primer, but I found that it
works fine to sequence with one of the same primers that were used for PCR.

	-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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