Protocol for Re-using of Qiagen tips
Mads Noerregaard-Madsen
5M NH(4)Ac, pH 9.5, then 3 volumes water and finally 2-3 volumes 96%
EtOH. Then place a paper towel on top of the tip and blow out residual
EtOH using pressured air (not necessary with miniprep columns), place
the tip at 42 degrees ON. The tips can be regenerated app. 5x.
The columns seems completely clean after the regeneration - at least
I've never had any problems with carrying over DNA from one prep to
another.
Mads Norregaard-Madsen, m...@biobase.dk
Department of Molecular Biology
Odense University
Denmark
Trevor Bezdek
Quiagen only recommed 50 ml of overnight culture for a maxi prep in order
to not overload the column? Also, if you are using TG1 it will increase
the problem.
In article <Do7KE...@uns.bris.ac.uk>, harry....@bristol.ac.uk wrote:
> 1) Too much bacterial material in starting material (I solubilize a
> 250 ml overnight culture of Terrific broth into 10 ml P1, then add 10 ml P2
> and P3).
--
Trevor Bezdek
Stanford Medical School
Developmental Biology
<tre...@leland.stanford.edu>
Harry J. Witchel
I routinely have a problem with Qiagen maxi preps in that the columns
flow very slowly, as if they were clogging. To run 30 mls through a column
might take 3-4 hours on a bad day. To reduce this problem, I have taken to
respinning the initial KOAc supernatant a second time a high speed, and then
to run the entire supernatant through a .45 um syringe filter before loading
onto the column. Even so, the column sometimes (unpredictably) slows down.
My two guesses would be that:
1) Too much bacterial material in starting material (I solubilize a
250 ml overnight culture of Terrific broth into 10 ml P1, then add 10 ml P2
and P3).
2) I let the column run dry after wetting it with QB. Usually I load
just when QB is running out, but sometimes there might be 10 minutes where the
top of the column is dry. Qiagen claims that their column design should
prevent the actual matrix itself from drying out.
Do you have any guesses?
Thanks,
Harry
In article <3144DC...@biobase.dk>, m...@BIOBASE.DKo says...
Ferland Louis H.
I would go for your guess #1. I had the same problem of too much muck in
the culture to generate the large quantities of some of my plasmids. I
solved the problem with the good old chloramphenicol amplification trick:
I inoculate 500 ml of LB in the morning, add the chloramphenicol (I
can't recall the concentration from the top of my head, but it was pretty
standard) when the OD at 660 nm is around 1.0-1.2 (usually 3.5 to 4 hours),
and let grow further overnight. That gives anywhere between 1 and 3 mg of
plasmid that can be purified on a Qiagen Mega column without any problem
of slow elution.
Hope this helps,
Louis
On Wed, 13 Mar 1996, Harry J. Witchel wrote:
> Date: Wed, 13 Mar 1996 13:35:07 GMT
> From: Harry J. Witchel <Harry....@bristol.ac.uk>
> To: met...@net.bio.net
> Subject: Re: Protocol for Re-using of Qiagen tips
>
> Mads --
> I routinely have a problem with Qiagen maxi preps in that the columns
> flow very slowly, as if they were clogging. To run 30 mls through a column
> might take 3-4 hours on a bad day. To reduce this problem, I have taken to
> respinning the initial KOAc supernatant a second time a high speed, and then
> to run the entire supernatant through a .45 um syringe filter before loading
> onto the column. Even so, the column sometimes (unpredictably) slows down.
> My two guesses would be that:
>
> 1) Too much bacterial material in starting material (I solubilize a
> 250 ml overnight culture of Terrific broth into 10 ml P1, then add 10 ml P2
> and P3).
>
> 2) I let the column run dry after wetting it with QB. Usually I load
> just when QB is running out, but sometimes there might be 10 minutes where the
> top of the column is dry. Qiagen claims that their column design should
> prevent the actual matrix itself from drying out.
>
> Do you have any guesses?
> Thanks,
> Harry
>
> Harry....@Bristol.Ac.Uk
>
>
Dr. Louis H. Ferland
Centre de Recherche, Hotel-Dieu de Montreal
Dept de Nutrition, Universite de Montreal
Phone: (514) 843-2757 FAX: (514) 843-2719
QIAGEN
In response to Dr. Witchel and Dr. Ferland......Guess number 1 below.
For the QIAGEN-tip 500 or "maxiprep" we recommend 100 ml of LB culture to
isolate 300 to 500 micrograms of plasmid (for a high copy plasmid expect
3-5 micrograms per ml LB). If you use rich broth, it is necessary to scale
back the culture volume as the cell density is 4-5 times that of LB broth.
A high cell density would require more alkaline lysis buffers (to avoid
"muck" and the resulting clogging or slow flow rates) and a larger
QIAGEN-tip to bind the higher amounts of plasmid DNA (e.g., Dr. Ferland's
recommendation蟻ssuming this a low copy plasmid, since it was
chloramphenicol amplified).
The recommended LB culture volumes in the QIAGEN Handbook do give the most
reproducible yields and highest DNA quality.
In response to Dr. Witchel.....Guess number 2 below.
QIAGEN-tips can be left unattended. The column will not dry out that
quickly. You could easily set up another experiment or go to lunch!
Hope this helps.
For more information or a free QIAGEN Plasmid Handbook, please contact
QIAGEN Technical Services.
Sincerely,
QIAGEN
In article <Pine.3.89.9603200...@mistral.ERE.UMontreal.CA>,
--
QIAGEN, Inc.
1-800-426-8157
Temporary e-mail address: qia...@Kaiwan.com
Ferland Louis H.
> Date: 22 Mar 1996 20:52:18 GMT
> From: QIAGEN <qia...@kaiwan.com>
> To: met...@net.bio.net
> Subject: Re: Protocol for Re-using of Qiagen tips
>=20
> Dear QIAGEN users,
>=20
> In response to Dr. Witchel and Dr. Ferland......Guess number 1 below.
>=20
> For the QIAGEN-tip 500 or "maxiprep" we recommend 100 ml of LB culture to
> isolate 300 to 500 micrograms of plasmid (for a high copy plasmid expect
> 3-5 micrograms per ml LB). If you use rich broth, it is necessary to scal=
e
> back the culture volume as the cell density is 4-5 times that of LB broth=
.
> A high cell density would require more alkaline lysis buffers (to avoid
> "muck" and the resulting clogging or slow flow rates) and a larger
> QIAGEN-tip to bind the higher amounts of plasmid DNA (e.g., Dr. Ferland's
> recommendation=8Bassuming this a low copy plasmid, since it was
> chloramphenicol amplified).=20
Actually, this was a pBlueScript-based construct, though I admit it does=20
not grow as well as the empty vector. This being said, since slow flow=20
rate of Qiagen columns occured in our hands with a number of constructs=20
(grown in XL-1 Blue, I have not had this with other strains), we now apply=
=20
the chloramphenicol for all=20
our plasmid preps (and I haven't used low copy plasmids in close to ten=20
years!). This is therefore not so much to have more plasmid copies per=20
bacteria, as to have less bacteria per ml while maintaining high plasmid=20
amounts per ml of culture.=20
Harry J. Witchel
Harry