Qiagen: This is ridiculous

Vladimir Svetlov vvsvetlov at utmem1.utmem.edu
Sun Aug 25 01:36:41 EST 1996


In article <4vno7a$2qu at panix.com>, iayork at panix.com (Ian A. York) wrote:

> I've been using Qiagen DNA preps for years.  The quality is great; as 
> everyone who's used them knows, though, the yield is at best 
> inconsistent.  At random intervals, and for no apparent reason, there 
> will be absolutely no DNA produced.  I've been willing to deal with that 
> when I was getting 5 out of 6, or whatever, with good yields; with 
> important DNA, I'd always run duplicates to be reasonably sure of getting 
> something.
It your right to be pissed but it might be more productive to find a
source of the problems in your protocol.
Unless no one else uses your reagents I'd start with finding a dumbass
with hands growing out his/her rear end that used the kit before - every
place has at least one. Like last week suddenly Genomyx kit stopped
sequencing while newly purchased fmol kept on rolling with the same
template and primers. Untill that is the same schmuck got his hands on it
too. Now if you can't find one... For all years we have been using Qiagen
kits I never had any problems with it except inconsistent flow rates on
gravity columns - the main reason we switched to vacuum-driven Promega
midi's. Once the entire batch was screwed up and was promptly replaced but
I never experienced yield inconsistencies you describe. I still use about
100-150 Qiaminis a month and can attest to their uniformity in yields. 

 
> In the last two days, I've run 10 Qiagen DNA preps - 8 midi-preps, two
> maxipreps - and got one (1) with detectable DNA.  Meanwhile two identical
> cultures, run on a different company's resin, gave 160 ug and 175 ug of
> DNA from 200 ml bugs (low copy-number plasmid). 

You probably used also other company's reagents - incl. NaOH for alkaline
lysis. In my experience it is the first one to "go" - people leave it open
on the table for few days and still think they (and everybody else) can
use it for lysis. Another reason why you are getting inconsistent yields
with low-copy number plasmids in Qiagen protocols might be the intrinsic
nuclease activity. Q-people say in the protocol that one can omit (and one
usually omits everything s/he can and then a bit more <G>) chaotropic PB
wash when working with low-nuclease hosts like DH5alpha. In some other
kits it is not an option since guanudinum salts are added to the resin
itself. And even with DH5alpha cells nucleolytic degradation is not out of
question. 
Now you can resume being pissed.
Regards,
VS
PS - whereever Kolya Chitaev can be found there are some weird bugs nearby.



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