erase-a-base

Bruce Roe broe at aardvark.ucs.uoknor.edu
Wed Jun 17 07:26:00 EST 1992


In article <1992Jun16.912.898 at channel1>, "michael coyne" <michael.coyne at channel1.com> writes...

	<stuff deleted>

>why she is unable to retain the DNA?  Anyone else encounter problems
>with this kit?
> 
>We thought that it might involve the plasmids being nicked
>(single-stranded) and therefore vunerable to exo III at several areas,
>but this does not seem to be the case.
> 
Mike,
	The kit's not the problem, the templated DNA is, based on this
and your previous posting.  It's my guess that the template IS NICKED
contrary to your previous statement.  Have her take some template DNA,
sequencing buffer, dNTPs (one of which is radio-labeled) and some enzyme
(Sequenase if you like) and do a 15 minute incubation without added primer.
Then run the products on a sequencing gel and any radioactivity she sees
will be due to the presence of internal nicks with free 3' hydroxyls that
can serve as internal primers for this reaction.  My guess is that the
gel will be hotter than h--- .
Good luck..........bruce
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