Qiagen: This is ridiculous

Ian A. York iayork at panix.com
Mon Sep 2 22:25:58 EST 1996


In article <322B8BAF.376E at unity.ncsu.edu>,
Susan Jane Hogarth  <sjhogart at unity.ncsu.edu> wrote:
>
>At the risk of starting a new set of "kit wars", I just am wondering if
>this is really worth all the trouble... was the yeild/purity using the
>"old" methods really that bad? No offense meant; I really don't know...

For subcloning, no problem; you can do pretty much anything you want so
long as you don't actually spit in the tubes.  For sequencing, you need
clean but not ultraclean DNA - most kits or good-quality minipreps will
do.  (This is for automated sequencing, I don't do my own.) For
transfecting mammalian cells, you need clean DNA; how clean depends on the
cells and the transfection protocol.  I've run into one or two cases where
Qiagen DNA gave good results but the DNA from another company gave a lot
of dead cells; but I've also used DNA from The Other Company's kits in the
same transfection protocol successfully. 

I haven't any experience with techniques like gel retardation and so
forth; my understanding is that transfection of mammalian cells tends to
be the most demanding use of DNA, so I imagine that most other kits would 
be okay for them.

I've rarely done cesium chloride DNA preps; they're considered the 'gold 
standard' as far as quality, and are high on the list as far as quantity 
is concerned.  Qiagen is about the same in quality, but gives less DNA 
(if you get any).  Other companies' kits - I have limited experience 
here, I've only tried a couple of others - seem to give larger amounts of 
DNA than Qiagen with slightly lower quality.  

It depends very heavily on what you want to do with it.  I'm fairly
transfection-oriented these days, which is why I've kept the Qiagen
around; if I was just subcloning or putting it into yeast, I'd stick with
The Other Company entirely, because it's cheaper and gives more DNA more 
consistently.  

I should note that there are a handful of restriction enzymes which are
very picky and which might work better given Qiagen-quality DNA.  For my
part, I've had no problems with DNA from The Other Company's kit and some
enzymes I've heard of as being somewhat fussy: KpnI, NdeI, and some others
- but some of my lab mates, using DNA from a LiCl technique that produces
a ton of rather grungy-looking DNA, have had trouble with them.  I don't
think that Qiagen quality is necessary for subcloning. 

Ian
-- 
      Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
      "-but as he was a York, I am rather inclined to suppose him a
       very respectable Man." -Jane Austen, The History of England



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