magic mp & sequenase

Bruce Roe broe at aardvark.ucs.uoknor.edu
Thu Dec 3 08:44:00 EST 1992


In article <1992Dec3.912.2122 at channel1>, "michael coyne" <michael.coyne at channel1.com> writes...
> 
>Hello all...
> 
>In advance of some experiments to be performed by a co-worker (and at
>the request of my boss), I would like to find out if anyone has tried
>using plasmid DNA recovered with Promega's Magic MiniPrep kit directly
>for use with the Sequenase kit.
> 
>The plasmid we're recovering is small (apx. 6 kb), yield and cleanliness
>look good on agarose gels, but the Sequenase kit has been (in our hands)
>somewhat fussy about the DNA used.
> 
>I remember, for example, a post here not too long ago discussing the use
>of alkaline-lysis (Birnboin & Doly) mini-prep DNA with the Sequenase
>kit, after cleaning it up with glassmilk.  This protocol, as I recall,
>advised using two times the recommended volumes of the Sequenase kit
>reagents when using this method.
> 
>Has anyone had similar experience tweaking the kit to accept plasmid DNA
>recovered with the Magic MiniPrep kit?
> 
>Any information as to the viability of this approach, and suggestions
>for its successful implementation, will be greatly appreciated.
> 
>Thanks...
> 
>Mike
>michael.coyne at channel1.com

Hi all,
	Having just read 2 very good messages about "kits" I then read
this one.  No flame intended Mike, but if you are going to be spending
your tax dollars and mine on a kit, the call the company and ask them 
the question.  I'm sure they have lots of experience in answering these
types of questions.
	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.
Because the quality of the template is less of a factor with end-labeled
primers, you should be able to eliminate the column purification step.
Cheers......bruce
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 /  University of Oklahoma     INTERNET: BROE at aardvark.ucs.uoknor.edu  \
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