magic mp & sequenase

wmelchior at ntet.nctr.fda.gov wmelchior at ntet.nctr.fda.gov
Thu Dec 3 10:31:34 EST 1992


> 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.
...
> Has anyone had similar experience tweaking the kit to accept plasmid DNA
> recovered with the Magic MiniPrep kit?

I also am curious about this.  I was recently given some samples of the 
Promega system, which claims to act "more effectively than silicon-based 
media" but is used in a manner VERY similiar to powdered silica, except it 
uses guanidine HCl instead of NaI.  (Both are chaotropic agents, so the 
binding mechanism is probably the same.)  I have not had a chance to try
the Magic Minipreps yet, but hope in the next couple of months to compare
them with the silica method that I posted here some time ago (described in 
the FAQ posting; it gives better results than anything else I've tried
except CsCl); I expect the Promega kit to give similar results with a
slight increase in ease and speed due its use of small columns for handling
of the matrix. (Using home-made silica powder, the silica method would be
cheaper and is probably not much more difficult.) 

I would appreciate any comments (especially negative ones) from anyone who
has tried it so I don't have to worry that I might be wasting my time.

(Sorry for rambling.  To answer the orignal question: I EXPECT the kit to 
work well with Sequenase.  I plan to merely increase the volumes of NaOH,
NaOAc and EtOH used for denaturation in the Sequenase protocol to handle
the higher volume produced by the kit, and then redissolve in the
USB-recommended amount of H2O after ethanol precipitation.) 
________________________________________________________________________________
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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