Reply to Fast& Cheap Plasmid Miniprep Kit
s535290 at aix1.uottawa.ca
Fri May 30 16:53:08 EST 1997
> 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.
I must also give my thumbs up to alkaline lysis/NH4Ac neutralization. It
is so fast and easy that I was wondering why people are willing to waste
their time using that "fast prep" method that people were talking
about, and which ends up requiring phenol extractions in the end.
I, however, did not know that the DNA from my quick preps was pure enough
for automated sequencing. I was always under the impression that it
wasn't. What system do you use ? we use ABI 373 with the dyedeoxy FS kits.
I always do preliminary minipreps using 200 ul of culture, check
for size, cutting with appropriate restriction enzymes, and then do
a quiagen prep of the remainder of the culture for the sole purpose of
automated sequencing. Don't tell me we've been wasting our money on
quiagen preps when the crude preps would have been ok !!!
For my (supervisor's) money, this is the best fast miniprep method out
there. The only limiting factor is minicentrifuge space, but such is the
case for every other method out there anyway.
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