Reply to Fast& Cheap Plasmid Miniprep Kit
bernard at elsie.nci.nih.gov
Thu May 29 13:16:40 EST 1997
In article <5mjucn$i24 at panix.com>, iayork at panix.com says...
>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.
>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.
Put me down as another vote for cheapy minipreps. I use the method
of Zhou et al. (Biotechniques, 8(2) 172-173 ) and the only
time it has ever failed for restriction digests is when I didn't
wash the pellet properly with 70% ethanol (and I've used a lot of
different enzymes in my time). Of course, I try to use endA- bacteria
(you have to add the phenol/chloroform step otherwise). If you think
you may get an ambiguous result then run some of the uncut preparation
beside the digested version (okay, so you have to double-comb the gel).
For sequencing I double-load (3 ml culture) and clean the
crude plasmid with PEG/NaCl which seems to work as well as phenol
but is cheaper and less nasty. This is for manual sequencing with
Sequenase so I don't know how automatics would cope. This fails so
seldom (does *anything* work 100% of the time!?) that I'll not
bother looking at anything else.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
(but soon moving to San Francisco)
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