direct sequenceing of lambda clone

William R. Morgan wmorgan at ACS.WOOSTER.EDU
Mon Sep 20 21:41:42 EST 1993


>Bruce Elder <elder at cgl.ucsf.edu> writes...
I am wondiering if anyone knows if it is possible to directly sequence
>a genomic DNA fragment in a lambda clone. I would be using primers directed
>to the genomic sequence, not the vector DNA. Any thoughts?
>
Yes, I have done this successfully (actually using a degenerate pool of
oligos!).  I followed an old protocol I received from New England Biolabs.

Very briefly, I first end-labelled 10 pmoles of the  sequencing primer with
T4 polynucleotide kinase and [gamma-32P] ATP (standard procedure).  After
alkaline denaturion (standard procedure), about 3 micrograms of the genomic
lambda clone was annealed with 1-3 pmoles of the end labelled primer (2
minutes at 65oC & cool to 35oC).  The primer was then extended with
Sequenase in the presence of 7.5 micromolar of each dNTP for 5 min. at room
temp. followed by the start Sequenase termination reactions.

I'm sorry this is so brief; if you're interested in pursuing this and would
appreciate a more detailed protocol let me know.

Bill Morgan
William R. Morgan
The College of Wooster
Department of Biology
931 College St.
Wooster, OH  44691
Phone:  216-263-2026
FAX:    216-263-2378
E-mail: wmorgan at acs.wooster.edu




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