magic mp & sequenaseDIR

wmelchior at ntet.nctr.fda.gov wmelchior at ntet.nctr.fda.gov
Thu Dec 3 12:06:53 EST 1992


> 	On the more positive side, sequencing off double stranded templates
> using radioactive incorporation can be troublesome if the templates are
> at all nicked. It's my guess that the variations between template
> preparations causing the sequencing reactions to be variable is directly
> related to the presence or absence of internal nicks.  You may want
> to try kinase labeling the primers with 32P since then the only product
> you'll see on your autoradiograph will be due to extensions from the
> radiolabeled primer and not from extensions from the nicked template.

Why wouldn't nicked templates cause noise by leading to premature 
termination (at nicks) of synthesis from primers?  I've been under the 
impression that avoiding this type of error was one of the advantages of 
dye-terminator chemistry for fluorescence sequencing:  You see only 
correctly terminated chains.

Responding directly to Bruce's suggestion:  The first time I used silica 
purification (sevral years ago), I got miserable sequences, which I
attributed to the nicking that takes place during this procedure.  But the
revised protocol I alluded to in my earlier message gives good results, so
either the newer method doesn't cause as much nicking or that wasn't really
the problem.  In either case, it's not necessary to avoid this type of 
purification just because you're doing ds sequencing.  (The silica method 
gives me good results both with 35S and with dye-terminators.)
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||     OMNISCIENCE
National Center for Toxicological Research   ||    Knowing what
Jefferson, AR  72079                         ||    thou knowest not
(501) 543-7206                               ||    is in a sense
                                             ||    omniscience.
WMELCHIOR at NTET.NCTR.FDA.GOV                  ||       from Grooks, Piet Hein



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