sequenase kit

Bruce Roe broe at aardvark.ucs.uoknor.edu
Wed Jun 17 07:13:00 EST 1992


In article <1992Jun16.912.897 at channel1>, "michael coyne" <michael.coyne at channel1.com> writes...
>Netters:
> 
>Though I have been following the discussion here regarding what seems to
>be fairly wide spread distaste with kits, I was asked by a colleauge
>(who doesn't have access to this net) to post the following question:
> 
>She is trying to sequence ds DNA using the Sequenase kit (USB).
>Her 1.9 kb insert is contained in an M13 plasmid vector.  She is
>preparing the DNA using large scale plasmid preps (alkline lysis) and
>purifying plasmid DNA through CsCl gradients.  She has also tried
>alkline lysis mini-preps and plasmid recovery using silica.  Her problem
>is that all she gets on the sequencing gel is a large smear.
> 
>Has anyone encountered a similar problem?  Any ideas as to where the
>problem might lie, or things to try to fix it?
> 
>Thanks...
> 
>Mike
>michael.coyne at channel1.com
> 
Mike,
	Double stranded templates are more difficult to sequence off of
than are single stranded templates.  There has been much discussion of
the "best" ways to isolate ds-templates and the conclusion I have reached
is all the methods work fine in the hands of an experienced individual.
In other words, her template is nicked, has too much salt, or is contaminated
with protein, genomic DNA and/or small RNAs.  She may want to try Prep-a-Gene
from BioRad as an additional clean-up step since only super-coiled DNA
seems to be adsorbed and eluted from this matrix and it may help.
	However, if she's already got the small insert in a "M13 plasmid
vector" - ?whatever that is?, why try to isolate ds-template? If she can
cut out the insert, put it into M13 and isolate ss-templates, or if it's
in a vector that will produce ss-DNA phage by super-infection, do that.
Single stranded templates give much better sequencing gels in the hands
of most individuals than do double stranded templates.
	Finally, if she questions the Sequenase Kit rather than the template
preparation, have her call USB, they are nice folks and will help her out.
Alternatively, try the BioRad Bst Kit (Bst = Bacillus Sterothermopholysis)
which gives much cleaner gels with either single or double stranded templates
in our hands.

Cheers......................bruce
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
 \  Bruce A. Roe               Professor of Chemistry and Biochemistry /
 /  University of Oklahoma     INTERNET: BROE at aardvark.ucs.uoknor.edu  \
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -  - - -
--Usual disclaimers - but if BioRad wants to send me some free reagents
  for giving them this plug, I'll be happy to save some $$ and not tell
  anyone :-)



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