Qiagen Maxiprep Problem
Benny Lo
the yield hasn't been too satisfactory. Recently, I found that when I let
the drop of ethanol that sticks on the side of tube evaporate off after IPA
precipitation and EtOH wash, a while precipitate forms where that droplet
sticks. This has led me to think that maybe the DNA has not been
sufficiently precipitated out of the solution. Is that a possibility?
I used 150mL of E coli culture and get only about 100 micrograms of plasmid
out. Given my experience described above, is there any way I can improve the
yield?
I also notice some white precipitate that has probably co-precipitated with
the DNA but will not redissolve on the addition of Tris, even after warming
to 37 degrees for a period of time. Very often I just have to spin it down
and take the supernatant as the DNA. Does anyone else experience this also?
Any advice will be appreciated. Many thanks.
Cornelius Krasel
> I have been using Qiagen Maxiprep to prepare plasmids for transfection and
> the yield hasn't been too satisfactory.
[...]
> I used 150mL of E coli culture and get only about 100 micrograms of plasmid
> out. Given my experience described above, is there any way I can improve the
> yield?
The yield very much depends on the kind of plasmid you use. With a
usual high-copy plasmid (Bluescript, pcDNA3 or the like) I get 600 ug
from 250 ml of culture.
I employ the following modifications:
1) Precipitate with isopropanol over night in the cold room.
2) Dissolve pellet in approx. 300 ul of water and reprecipitate with
2.5-3 vol EtOH in an Eppendorf tube. You should see the DNA
precipitate in the tube as soon as you add the EtOH and shake the
tube a bit.
3) Don't wash the resulting pellet with 70% EtOH.
4) Re-dissolve pellet by shaking for a few hours at room temperature.
The pellet is very difficult to dissolve, especially if it is
overdried.
HTH,
--Cornelius.
--
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany email: pha...@rzbox.uni-wuerzburg.de SP4 */
/* "Science is the game we play with God to find out what His rules are." */
Burkhard Hassel
<be...@holmes.nott.ac.uk> wrote:
>I have been using Qiagen Maxiprep to prepare plasmids for transfection and
>the yield hasn't been too satisfactory. Recently, I found that when I let
>the drop of ethanol that sticks on the side of tube evaporate off after IPA
>precipitation and EtOH wash, a while precipitate forms where that droplet
>sticks. This has led me to think that maybe the DNA has not been
>sufficiently precipitated out of the solution. Is that a possibility?
I think, the white stuff can be salts, not DNA. DNA precipitates
allmost instantaneously when 2-Propanol is added.
>I used 150mL of E coli culture and get only about 100 micrograms of plasmid
>out. Given my experience described above, is there any way I can improve the
>yield?
Depends on the vector and growth conditions. In order to evaluate the
yield, take 1 or two eppis of the culture and do a standard miniprep
(without columns) to see if it has something to do with Qia or not.
BTW, as in some labs the midi-kit is called maxi (cause maxis are
rarely used), did you really use the maxi?
>I also notice some white precipitate that has probably co-precipitated with
>the DNA but will not redissolve on the addition of Tris, even after warming
>to 37 degrees for a period of time. Very often I just have to spin it down
>and take the supernatant as the DNA. Does anyone else experience this also?
The same. I talked to the guys at Qiagen and even send them a probe of
this. They weren't able to identify what it was, but said it _could_
be protein-DNA complexes. Spinning it down is o.k.
In order not to overdry, I allways do the following: after
centrifugating the 70% EtOH wash, I suck the liquit of with a pasteur
pipette on a vacuum pump. Then I centrifuge down residual droplets
with a 1 min run. The remaining liquid I take of with pipette-man
(yellow tip). It's allmost impossible to suck in the pellet, don't be
affraid of this. Then I _don't dry_ and add the TE buffer. You can
watch the pellet dissolving within 1-2 secs.
Yours,
Bu.
Dave Bates
I have a slightly different problem. After nearly eight years of doing Qiagen
preps with absolutely no problem, about three months ago they stopped working.
All of a sudden we're getting no - or very little (<100ug) back. Qiagen haven't
been able to do anything other than send us a new kit - which worked for the
first two, and then the rest haven't worked again. We've done the diagnositics
and can't find the DNA anywhere-not in the washes, the isoprop, or anything.
Everything looks OK. These are kits in the UK.
Has anyone else had these sort of problems here in Britian? I'm beginning to
develop conspiracy theories
Dave Bates
--
Dr David Bates, Ph.D. Tel 0117 928 7823
Department of Physiology Mobile 0776 965 3923
The New Vet School Fax 0117 925 4794
Southwell Street email: Dave....@bris.ac.uk
University of Bristol
Bristol BS2 8EJ
Ski, golf, drink, eat. Life is sweet
http://www.bris.ac.uk/Depts/Physiology/Staff/DOB/dob1.html
Steve Cohen
yield drops to zero. You would think that the solid matrix would just
become saturated with DNA, but this is not the case.
This was a big topic here about a year ago, debate went on for months.
Steve
---
Chris LaRosa
Cornelius Krasel wrote:
>
The first problem I have with Qiagen is the price.....
There is Diatomaceous earth from Supelco (Celite 45) that works for
about 1/20 of the price and gives very good consistant results.
Basically the same thing as the also overpriced Prep-A-Gene for Bio-Rad.
Both companies offer fine products I found in general, but at exorbedent
prices.
PC LaRosa
Malay
Malay
Malay Kumar Basu
Centre for Cellular and Molecular Biology
Hyderabad 500007
I N D I A
Fax: (00-91)40-7171195
Phone: (00-91)40-7172241
-----
Cataholic: Can't stop bringing cats home.
-----
curi...@ccmb.ap.nic.in
----- Original Message -----
From: "Nokwanda Makunga" <mak...@botany.unp.ac.za>
To: <met...@hgmp.mrc.ac.uk>
Sent: Friday, April 07, 2000 12:56 AM
Subject: Re: Qiagen Maxiprep Problem
> We have a slightly different problem
>
> We have just tried to use our Qiagen kit and the little DNA we isolated
was degraded. The kit is about a year and half years old but has never been
used. We contacted the agent here in South Africa about the shelf-life and
they were clueless. Does anyone know how long one can keep these kits prior
to use ? Is it possible to just re-make the buffers and then just use their
columns as we cannot afford to buy a new kit at the moment.
>
> Nokwanda and Sara
>
> Research Centre for Plant Growth and Development
> School of Botany and Zoology
> Univeristy of Natal
> Pietermaritzburg
>
>
> ---
---
Wolfgang Schechinger
Reads very interesting. How did you find that solution?
The kit I've had problems with had been on the shelf for at least one
year. What might be the reason for that phenomenon? Is it CO2 that
diffuses through the plastic bottles and neutralizes the buffer?
Did you ever check the pH of a new and an older bottle?
Wolfgang
-----
This message is encrypted. Use your brain to decode it.
-----
Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
University of Tuebingen, Germany
email: wolfgang.s...@med.uni-tuebingen.de
wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
-----
usual disclaimers apply
-----
---
Malay
> Malay,
>
> Reads very interesting. How did you find that solution?
> The kit I've had problems with had been on the shelf for at least one
> year. What might be the reason for that phenomenon? Is it CO2 that
> diffuses through the plastic bottles and neutralizes the buffer?
> Did you ever check the pH of a new and an older bottle?
>
> Wolfgang
>
> > Check the elute buffer pH. Make it alkaline. It works.
> >
> > Malay
> >
I am sorry. I was replying to the post that complained about very low
yield. Can't say anything about shelf life but elution problem in Qiagen kit
is most of the time due to pH problem of elution buffer. Make sure that it
is slightly alkaline.
Malay
---