Reply to Fast& Cheap Plasmid Miniprep Kit

[Ian A. York]

In article <5miggm$1tck$1 at piglet.cc.uic.edu>,
Victor Levenson <levenson at uic.edu> wrote:
>
>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,

There are as many protocols for fast minipreps as, um, something that
there's a lot of, but in my experience all of them (including the NH4Ac
neutralization) are inconsistent.  That is, some work beautifully in one
lab but not another, some work 90% of the time but yield uncuttable DNA
the other 10%, and so on. 

The only way I found to get consistently cuttable DNA out of minipreps was
to include a phenol-chloroform step in the protocol.  If I did that, then
all the enzymes I used would cut; if I didn't, then 10% of the time or so
they wouldn't cut well.  

I use kits now, because I find that I get consistently cuttable DNA
without having to guddle around with phenol (and because we can afford
them); but even with kits I'll occasionally run into poor cutting.  I've
always been able to see the appropriate band, but it's not unusual to see
partial cuts as well; the partials aren't there when I do (clean)
large-scale preps.  

As far as I'm concerned, you can either do a phenol-chloroform extraction
and get clean miniprep DNA, or you can skip that and be careful to pick
enzymes that are good cutters.  

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