Maxiprep woes
Susanne Rohrer
I really hate them. We have tried qiagen, macherey-nagel, both are the
same. What bugs me is how expensive they are. This time I even wasted a
maxi column.
So I've talked to the rep before. First he said to lower the culture
volume (16 instead of 30 ml for a midi), then we find out you're not
supposed to use an overnight culture because the cells are already
starving and chewing up their own plasmids. on the other hand, they tell
you no, you can't use TB.
It just seems to me ages ago they used to work fine. I used to get like
300 ug from a maxi. What (the =*%+) is going on, and what are my
options?!
I mean, I get higher yields from a miniprep! Why can't they make their
hydrophobic matrix into maxi columns?!
Susanne in a state of agony...
Nick Theodorakis
I used to get odd ball low yields on Qiagen maxicolumns for no apparent
reason, too, and what really used to bug me was I know that the same
plasmid and same bugs used to give me more than a milligram from 50 ml
superbroth after CsCl purification. (Overnight culture, BTW, which is why
I doubt the "bugs chewed up my plasmid" excuse).
FWIW, I've seemd to have better consistency with BRL's "Concert" maxis
(The "high purity" ones, anyway).
Can you save some of the "pre-column" plasmid? Just take a half a ml or
so before the column, phenol extract, EtOH ppt. like you are doing a
miniprep, and run some on gel just to see if you have plasmid (and
estimate the pre-column yield). If you could show that there was plasmid
in the prep before you put it on the column,that would indicate a problem
with the column and not with the prep.
Nick
--
_______________________________________________
Nick Theodorakis
nicholas_t...@urmc.rochester.edu
Sent via Deja.com http://www.deja.com/
Before you buy.
Ian A. York
Susanne Rohrer <roger...@rzu-mailhost.unizh.ch> wrote:
>
>It just seems to me ages ago they used to work fine. I used to get like
>300 ug from a maxi. What (the =*%+) is going on, and what are my
>options?!
You're not alone; intermittent low yields from maxipreps is a chronic
problem in virtually every lab I've talked to. Qiagen columns are the
usual culprit, though to be fair they're also the most widely used.
I haven't had a problem with this for a long time now (knock wood), but I
don't know if it's something I've done or if I'm merely lucky--that's the
problem with intermittent problems.
Some things that are worth trying--none of which are guarnateted, or even
necessarily real concerns; there's a little superstition about this:
(1) Use LB rather than TB. For me, at least, it's more predictable. For
high-copy plasmids (e.g. pUC and derivatives) try 100 ml overnight
culture; for low-copy plasmids, try 200 or 250 ml.
(2) Don't use the QiaFilter system. I think that works well when there's
just the right amount of bugs, but it seemed much less consistent to
me. I use the centrifuge to spin out the crud.
(3) Try hard to avoid getting any little pieces of crud into the
column. Usually after a spin there's a good solid pellet, with a few
little flakes floating on top. I pour this into the column through a
piece of gauze, which filters out the last little pieces.
(4) If the solution you have after the spin is not clear--if it's cloudy
at all--pour it right back out of the column. I usually run this through
the Qiafilter, just as a final cleanup.
(5) Make sure you're recovering all the DNA from the final isopronanol
spin. At least half of it often sticks quite high up on the tube and can
be missed if you're only looking for a pellet.
Good luck.
Ian
--
Ian York (iay...@panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
Martin Chan
"Susanne Rohrer" <"srohrer(removethis)"@immv.unizh.ch> 撰寫於郵件
news:39F589DA...@immv.unizh.ch...
> For the n-th time I have done a Plasmid prep and got a really low yield.
>
> I really hate them. We have tried qiagen
I feel very happy using Qiagen QIAfilter midi system with described yield,
i.e. ~100ug plasmid. One of the tips is to give more time (except the lysis
step) than that mentioned in the protocol. When the manual says "incubate
for 10 min", give 30 min instead, etc.
Michael L. Sullivan
>(3) Try hard to avoid getting any little pieces of crud into the
>column. Usually after a spin there's a good solid pellet, with a few
>little flakes floating on top. I pour this into the column through a
>piece of gauze, which filters out the last little pieces.
>
I've also found that Miracloth (Calbiochem?) works great for this, if you
happen to have any around.
Mike
Michael L. Sullivan, Ph.D
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706
(608) 264-5144 Phone
(608) 264)-5147 Fax
---
Cornelius Krasel
> For the n-th time I have done a Plasmid prep and got a really low yield.
>
> same. What bugs me is how expensive they are. This time I even wasted a
> maxi column.
> So I've talked to the rep before. First he said to lower the culture
> volume (16 instead of 30 ml for a midi), then we find out you're not
> supposed to use an overnight culture because the cells are already
> starving and chewing up their own plasmids. on the other hand, they tell
> you no, you can't use TB.
>
> 300 ug from a maxi. What (the =*%+) is going on, and what are my
> options?!
1) Use a high-copy plasmid. With pcDNA3-based plasmids, we get more than
1 mg of DNA out of a Qiagen maxi column from a 250 ml culture which
is probably the capacity limit of the column. The culture volume could
probably be reduced much further, but I am too lazy to test this.
2) For Qiagen: do the precipitation step at 4°C over night. For the
subsequent centrifugation step, use 50 ml Falcon tubes in a swingout
rotor; we centrifuge at 4500 rpm (approx. 3800 x g) in a Hettich
Rotanta 96R. This is sufficient to pellet the DNA, and because of
the swingout rotor, it can be easily collected at the bottom of
the Falcon tube. (I used to do this with Corex tubes in a SS34
equivalent before. Switching to the method outlined here I increased
my yield by at least 50%.)
3) Don't wash with 70% ethanol.
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." */
biogeekgrrl
Yours is a common problem, don't fear. You've already lots of good
advice from the kind folks on this newsgroup. I'll just mention one or
two points which have, in my experience, proven to be important.
First, make sure you're getting complete lysis of your bacterial
pellet. If you have a 250ml overnight culture and spin all of it down,
try resuspending in twice the recommended amount of P1, and then double
the vols of P2 and P3. This has corrected the problem 85% of the time
in my experience.
Second, as has been mentioned, make sure you don't get any cell debris
in your Qiagen column. This generally mucks things up. I always run the
supernatant through a piece of gauze or filter paper before loading it
onto the column.
Other minor points: if you have a big plasmid ie 10kb-ish, these preps
are more troublesome. Try warming the elution buffer. Also, the pellet
after isopropanol precipitation tends to slip around the bottom of the
50ml tube. We always gently pipet off most of the isopropanol, then
transfer the pellet and remaining alcohol to a microfuge tube. It's
easier to remove the rest of the alcohol and do a 70% wash in these
tubes.
Hope this helps.
--
"Like most scientists, Gabe was oblivious to the fact that no one gave
a rat's ass about research unless it could be expressed in terms of
dollars."
-Christopher Moore
Alan Smith
3) Don't wash with 70% ethanol.
How can not washing with 70% ethanol increase the yield of a DNA prep? I
was under the impression that DNA was not soluble in 70%. Sometimes I wash
samples twice with 70% ethanol and have never noticed a loss in expected
yield. I have done about 25 qiagen midipreps and only had a problem when I
overloaded the with DNA, judged by looking at the supernatant from different
steps after the failure. Maxipreps might however behave differently.
Alan Smith
---
Arnoud van Vliet
recently I actually had one who confessed not to know anything about the
subject I was querying about, but would send me all the info I required :)
Coming to your problem, I can only suggest some things:
- spin the isoprop precicpitation in microcentrifuge tubes at full speed.
What I usually did with the midiprep: 5 ml eluate, add 4 ml isopropanol,
mix, fill 3 eps with 1.5 ml mix, spin 15 min full speed, decant supernatant,
add remaining mix to the three tubes, spin 30 min, etc. Works much better
than spinning in the big (usually dirty) tubes.
- I always used 100 ml LB overnight culture (pUC plasmids) per midi column.
Worked fine. The comments about that in all the Qiagen booklets are pure
rubbish! We know use the Qiaprep spin columns, and there you should use 3 ml
culture. We use up to 10 ml without problems.
- Keep as far away as you can from the &%$# QiaFilter. Ritually burn them
(together with the dreadful QiaEx II kit)
hope this helps
Arnoud
"Susanne Rohrer" <"srohrer(removethis)"@immv.unizh.ch> wrote in message
news:39F589DA...@immv.unizh.ch...
> For the n-th time I have done a Plasmid prep and got a really low yield.
>
> I really hate them. We have tried qiagen, macherey-nagel, both are the
> same. What bugs me is how expensive they are. This time I even wasted a
> maxi column.
> So I've talked to the rep before. First he said to lower the culture
> volume (16 instead of 30 ml for a midi), then we find out you're not
> supposed to use an overnight culture because the cells are already
> starving and chewing up their own plasmids. on the other hand, they tell
> you no, you can't use TB.
>
> It just seems to me ages ago they used to work fine. I used to get like
> 300 ug from a maxi. What (the =*%+) is going on, and what are my
> options?!
Ian A. York
Arnoud van Vliet <avv...@knoware.nl> wrote:
>Some people still talk with the reps? Gave up a long time ago, although
>recently I actually had one who confessed not to know anything about the
>subject I was querying about, but would send me all the info I required :)
The three Qiagen reps I've dealt with over the years have been either very
good, or excellent: not only for technical knowledge and troubleshooting
ability, but for things like notifying us of good deals, providing
samples, and general salesmanship stuff. I'd have dropped Qiagen
altogether several years ago if it wasn't for one of their reps.
Cornelius Krasel
> Cornelius Krasel wrote:
>> 3) Don't wash with 70% ethanol.
>
> How can not washing with 70% ethanol increase the yield of a DNA prep?
I have often observed that 70% ethanol decreased the yield of a DNA prep.
I don't think that it is because of the DNA being soluble in the ethanol,
but because the pelleting of the DNA after the wash is incomplete.
Hartley, Jim
on the column. Precipitate with 2 vols of ethanol or 1 vol isopropanol,
dissolve in TE, apply to gel next to supercoiled stds. Apply what you
recover from the column in an adjacent lane. Some crude calculations of the
volumes will tell you whether your losses came before or after the column.
Do you need to use a purification column? They may remove some protein,
although there is almost none to begin with, and most, but not all, of the
RNA (really overload a gel and you'll likely see some). But they don't
remove chromosomal or nicked or dimer plasmid as far as I can tell. Perhaps
you should consider direct alcohol precipitation of the cleared lysate.
FWIW, all the standard cloning E. coli strains are endA minus, which means
that phenol extraction of alkaline lysis DNA is unnecessary, at least to
protect the DNA. Try doing a RE digestion with and without a phenol
extraction.
Jim Hartley
James L. Hartley, Ph.D.
Life Technologies / Invitrogen
PO Box 6482
9800 Medical Center Drive
Rockville, MD 20849-6482
Tel. 301-610-8337
Fax 301-610-8371
jhar...@lifetech.com
---
Student
plasmids I want to purify are low-copy number ones. I did however sort out a
compromising solution that leads to about 200-250 micrograms of DNA from a
360mL culture.
What I do is this:
1) Spin 2x160mLs of bugs in LB separately and treat as separate samples
during resuspension, lysis and netralization and filtering with Qiafilter.
2) Filter the two lots of supernatants into ONE Qiagen column and proceed as
normal
3) Spin for 1 hour, and 30 mins with IPA and 70% EtOH respectively rather
than the recommended 30mins and 15mins.
This may be a costly solution but I can't lose much time optimising the damn
thing.
Hope this helps.