Reply to Fast& Cheap Plasmid Miniprep Kit
kraev at bc.biol.ethz.ch
Wed Jun 4 08:34:34 EST 1997
In article <5miggm$1tck$1 at piglet.cc.uic.edu>, levenson at uic.edu (Victor
> Qguo at aol.com wrote:
> >I have used many different commercial kit for Plasmid miniprep, usually
> >Quagen's plasmid Purification kit worked well, it worked stable, and fast.
> >But recently I am using Omega Biotek's miniprep kit, it works very good, get
> >very good yield as Quagen, it is a little bit fast, one miniprep takes only
> >10 mins, average yield is about 30mg, the most important reason for me to use
> >this kit is that it is much cheap: only $35 for 50 minipreps. I think it is a
> >good deal. I hope you call them to asking a free sample or visit their Web
> >Page: www. Omegabiotek.com. I hope this information can help you at some
> >point. Good luck for your experiment.
> I don't want to spoil anyone's business ;-) and to re-ignite
> "anti-kit" polemics (where are you, Jim?), but why, for crying out
> loud, are you guys spending all that time AND money when alkiline
> lysis with NH4Ac neutralization gives such a quick and wonderfully
> pure miniprep? Yes, it is good enough for automatic sequencing and NO,
> it is not good enough for transfection. 18 minis take about 45 - 60
> min and you get up to 2 ug plasmid from 1.5 ml of o/n culture. I have
> not calculated the cost per miniprep (2 epp tubes are the most
> expensive items, I believe), but it surely beats ANY kit.
So you are ( and all of this thread up to now) into the "alkaline" philosophy.
Here comes something from the "boiling" one. Why are you guys spending
some much time and money .. (sounds familiar?). My favourite is a modification
of the boiling lysis, published in 1985 in a small TIBS article. You make
2.5 M LiCl and the ribosomal RNA goes into pellet , and you do not need
RNAse (I feel safer when I prep RNA and my student preps plasmids:-)), because
the remaining low MW RNA does not interfere with anything
downstream, except with seeing very small fragments on digests. Other reasons
why I use it are: the TELT solution does NOT go bad on the shelf for a
year or so,
and it is the only solution you need (and lysozyme). As for the
cleanup, you have
the choice between the chloroform and a resin called Advamax (AGTC Corp.).
Additional benefit is that the size can be checked before the cleanup and only
clones destined for (yes automated) sequencing are cleaned. I failed to use the
package of Advamax before it dryes up, actually, and had to aliquot it
tubes... <Most of the time> the preps before cleanup can also be cut with
if the particular clone yield is normal.
There was an interesting comment that in the South they use more kits than
in the other
parts of the USA. Before someone objected: well, Qiagen kits with their
compensate for the lack of proper education...but then you have no chances
of turning the
kit addicts into your "religion" (neither do I, in fact ;-).
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
e-mail kraev at bc.biol.ethz.ch
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