Qiagen: This is ...
qiagen at kaiwan.com
Wed Sep 4 09:28:54 EST 1996
In article <322095B4.6D83 at visar.wustl.edu>, nikolaic at visar.wustl.edu wrote:
>>To: methods at net.bio.net
>>From: iayork at panix.com (Ian A. York)
>>Subject: Qiagen: This is ridiculous
>>Date: 24 Aug 1996 16:20:58 -0400
>>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
>>Blowing off steam,
>What you describe sound very familiar.
>The similar things happend in our lab about 2 yars ago. Apparently, at no
obvious reason mini-preps will yield no DNA, using Qiagen. As it turned out,
there is nothing to do with the Qiagen kits at all. It was the bugs!
We switched from Fisher`s amp. to Sigma`s. Discarded all our plates, clearned
the place, water baths and etc. Took the fresh frozen stock of E.coli,
prepared a competent cells using new antibiotic, and you guess it: problem
dissapiared. All 100% of mini- or midi- or max- or whatever preps using
Qiagen start working again with 100% yield. "Its very difficult to find a
grey cat in dark room, if she isn`t there"
In article <9608038417.AA841785024 at HRI.BBSRC.AC.UK>, trevor fenning
<trevor.fenning at hri.ac.uk> wrote:
>PS Why don't Qaigen answer all these queries themselves ? Don't they read
what everyone is saying about them on the Bionet ?
Recently, several subscribers to this newsgroup have commented on their
experiences with QIAGEN Plasmid Kits. QIAGEN appreciates that the Bionet
methods and reagents newsgroup is a forum for users to discuss both
homemade methods and commercial kits such as ours. We are hesitant to
enter a discussion of our products too early as we are interested in
hearing the opinions of our customers. However, at the same time, we would
like to avoid any misunderstandings concerning our products. In addition,
we would be happy to provide (nonproprietary) information when asked.
QIAGEN Plasmid Kits are subject to strict quality control to ensure their
quality and reproducibility. Unfortunately, the quality of the bacterial
culture which forms the starting material for all plasmid purifications
depends on several variables, and may not always be reproducible. QIAGEN
has developed a series of recommendations designed to minimize the
variables in the starting culture which can affect the quality and
reproducibility of the plasmid preparation.
Following our culture volume recommendations will normally prevent most
problems with inconsistent or reduced quality results. Please review the
culture volume recommendations for the kit you are using. As Nikolai
states above, good microbiological practice is important, too (see
previous message in thread from Tsichlis Lab at Fox Chase Cancer Center.
When they replaced their XL-1 stock, their plasmid isolation problems went
away). We recommend growing cultures in LB media and harvesting during
early stationary phase (12-16 hours). Rich media will give less consistent
results as cultures reach a higher cell density and the stationary phase
much earlier than cultures grown in LB media. Cultures grown in rich media
should be harvested between 8-10 hours, otherwise there may be a
significant percentage of cells that have lysed or lost the plasmid due to
depletion of antibiotic in the media. The presence of these cells will
reduce the quality of the purified DNA.
Buffers may function poorly if they have been contaminated or stored
incorrectly. They are simple buffers which can easily be prepared fresh,
if necessary, from recipes provided in the QIAGEN Plasmid Handbook.
Inconsistent results can also occur if the lysis buffers are not well
mixed, or if precipitate is carried over onto the columns. Ensure that the
lysate is centrifuged well (preferably 2X) or filtered through QIAfilter
to avoid any problems. Transfer the cleared lysate to the column soon
after clearing to prevent any additional precipitate from forming.
If you do experience problems, the easiest way to confirm that plasmid DNA
was initially present, and to tell what happened to it, is to save all
fractions (cleared lysate, flow-through, wash, eluate). If the plasmid is
missing, you can determine precisely what happened by precipitating a
small portion of each fraction with isopropanol and running it on an
agarose gel. If the plasmid was present in the initial lysate, it can be
recovered from one of these fractions, and repurified once the cause of
the problem is diagnosed. A complete protocol for this may also be found
in the QIAGEN Plasmid Handbook which accompanies every kit.
QIAGEN is committed to providing high quality products. All QIAGEN
products are backed by stringent quality control procedures and 100%
satisfaction. If any product fails to meet your expectations, please call
our Technical Service Group for help or for more information.
Temporary e-mail address: qiagen at Kaiwan.com
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