Minipreps and Automated Sequencing
Christopher Walentas
(mini-prep scale) for ABI automated sequencing? We've tried Promega Wizards
(Magic uesd to work but that's another story), but they don't like to yield
any sequence even with additional precipitations/ washed to remove excess salt.
We tried QIAGEN 20 tips-- they work great down the hall where they use pUC
and cosmids, but the yields for pKK223-3 and pGP1-2 derived vectors are
deplorable. Folk here have even put CsCl banded DNA down a 20 tip to find
that they are only able to recover 10% or so. What we've settled on is either doing larger minpreps (or rather a bunch of 'em), or chloramphenicol. Is there a better way?
As an aside, if Promega was in patent conflict with their Magic resin, who
DOES have the patent so that I can go back to what used to work.
Please forward any and all suggestion to the email address below.
Cheers in advance!
Tripp
-
Christopher D. Walentas
Centre for Molecular Recognition, Tel.:
Sir William Hardy Bldg., within the U.K. (0223) 333662
Department of Biochemistry, outside +44 (223) 333662
University of Cambridge, FAX 333345
Tennis Court Road, Answerphone & alt. FAX 333661
Cambridge, England CB2 1QW
e-mail: c.d.wa...@bioc.cam.ac.uk
under the supervision of Professor R. N. Perham
Brian Margolin
: Does anyone out there know what the best way to prepare low-copy number plasmid
: (mini-prep scale) for ABI automated sequencing? We've tried Promega Wizards
: (Magic uesd to work but that's another story), but they don't like to yield
: any sequence even with additional precipitations/ washed to remove excess salt.
: We tried QIAGEN 20 tips-- they work great down the hall where they use pUC
: and cosmids, but the yields for pKK223-3 and pGP1-2 derived vectors are
: deplorable. Folk here have even put CsCl banded DNA down a 20 tip to find
: that they are only able to recover 10% or so. What we've settled on is either doing larger minpreps (or rather a bunch of 'em), or chloramphenicol. Is there a better way?
Promega is now selling a wizard kit, called the Wizard 373 DNA
Purification System, specifically designed for prepping plasmid DNA to use
in an ABI sequencer. They are giving away free samples. I just got my
free sample, but have not tried it yet. It seems that it is just the
Wizard miniprep kit with an additional Centricon purification step at the
end.
------------------------------------------------------------------------------
Brian Margolin, PP-ASEL-IA marg...@darkwing.uoregon.edu
Institute of Molecular Biology (503)346-5197
University of Oregon
Eugene, OR 97405
Amos H Heckendorf
: Does anyone out there know what the best way to prepare low-copy number plasmid
: (mini-prep scale) for ABI automated sequencing? We've tried Promega Wizards
: (Magic uesd to work but that's another story), but they don't like to yield
: any sequence even with additional precipitations/ washed to remove excess salt.
: We tried QIAGEN 20 tips-- they work great down the hall where they use pUC
: and cosmids, but the yields for pKK223-3 and pGP1-2 derived vectors are
: deplorable. Folk here have even put CsCl banded DNA down a 20 tip to find
: that they are only able to recover 10% or so. What we've settled on is either doing larger minpreps (or rather a bunch of 'em), or chloramphenicol. Is there a better way?
: Christopher D. Walentas
: Centre for Molecular Recognition, Tel.:
: Sir William Hardy Bldg., within the U.K. (0223) 333662
: Department of Biochemistry, outside +44 (223) 333662
: University of Cambridge, FAX 333345
: Tennis Court Road, Answerphone & alt. FAX 333661
: Cambridge, England CB2 1QW
: e-mail: c.d.wa...@bioc.cam.ac.uk
: under the supervision of Professor R. N. Perham
We have found that with and IPTG-induced 1 liter culture of P1 the yields
on Macherey-Nagels competing product(see relationship below) are
typically greater than 300ug. the attached protocol may be helpful to
you. While the products are not identical (different silica, spacer arm
for the surface chemistry, and functional group) this may help you get
more yield. If it doesn't then Nucleobond is available though a
distributor in the UK.
Please respect the copywrite. C 1995 Dr. Kevin Doyle for The Nest Group, Inc.
The Nest Group, Inc. 45 Valley Road, Southborough, MA, USA 508-481-6223
FAX# 508-485-5736 email: nes...@world.std.com
Nucleobondª Technical Notes
Number 5.1.2-5
** Special Conditions When Using Nucleobond AX to Purify P1 Constructs and
Other Low Copy Number Plasmids and Cosmids
When isolating P1 constructs and other very low copy number plasmids
using the Nucleobond AX alkaline lysis-based protocol, the potential
always exists for lower than expected yields to be obtained. While, in
general, there are a number of factors that could adversely affect
plasmid yield (most of which are addressed in the Nucleobond AX
Properties and Applications Guide ), in the case of low copy number
plasmids, incomplete bacterial lysis is the number one culprit. The
primary reason for this is that in order to get the maximum yield for a
particular cartridge size, many researchers grow cultures of these
plasmids that are larger than can be efficiently handled by the
prescribed volumes of lysis buffers for the cartridge. The first and
most obvious solution is to increase the amounts of the lysis buffers to
be used in these cases. However, how much is sufficient for a particular
culture? Are there any other ways to alleviate the incomplete lysis problem?
(A) Some Golden Rules for Lysis Buffer Volumes
Whether the copy number of your plasmid is high or low, there are
certain simple rules regarding the minimum volumes of the lysis buffers,
S1, S2, and S3, to be used. These are as follows:
(1) Low Copy Number Plasmids That Have Not Been CAPped or P1
Constructs Whether or Not They Have Been Induced (see below)
Use a minimum of 4.0 ml of each lysis buffer per 100 ml of culture
regardless of which cartridge size is being use. However, if the
particular cartridge requires more than this, then use the prescribed
amount for the cartridge. For example, if a 100 ml non-CAPped culture
of a low copy plasmid is to be processed on an AX-500 cartridge then
use the prescribed 12 ml of S1, S2, and S3 for the cartridge. On the
other hand, if a 500 ml non-CAPped culture is to be processed on an
AX-500 cartridge then at least 20 ml ({500 ml / 100 ml} * 4) of each of
the three buffers should be used according to this rule to insure
proper cell lysis.
(2) High Copy Number Plasmids or Low Copy Number Plasmid That Have
Been CAPped (see below)
Use a minimum of 1.0 ml of each lysis buffer per 100 ml of culture
regardless of which cartridge size is being use. As in (1) above, if
the particular cartridge requires more than this then use the
prescribed amount for the cartridge. For example, if a 100 ml CAPped
culture of a low copy plasmid is to be processed on an AX-500 cartridge
then use the prescribed 12 ml of S1, S2, and S3 for the cartridge. On
the other hand, if a 500 ml CAPped culture of a low copy number is to
be processed on an AX-500 cartridge then one would still use the
prescribed 12 ml of S1, S2, and S3 for the cartridge according to this
rule to insure proper lysis.
(B) Culture "CAPping" and Induction
Years ago before the advent of today's extremely high copy number
plasmids, cultures were routinely CAPped with an antibiotic to which the
bacteria with plasmid were not resistant. CAPping shuts down the
protein production machinery of the cells, but permits the plasmid DNA
to continue to replicate, thus increasing its copy number. CAPping is
not done with high copy number plasmids because it is not needed and
probably would have little if any effect, anyway.
Chloramphenicol dissolved in ethanol is probably the most often used
antibiotic for CAPping usually in the concentration of 150-170 µg/ml of
culture. Other antibiotics like spectinomysin are used when the plasmid
confers chloramphenicol resistance.
Induction is done on cultures of P1 plasmids with the same purpose as
CAPping, that is to increase copy number. While similar in concept at
least to CAPping, it does not require antibiotics. Instead, treating a
P1 culture with IPTG can effectively increase copy number from 1 to
about 20. Unlike CAPping, P1 cultures that have been induced should
still be handled following rule (1) above.
In cases were CAPping or induction is planned on being used, smaller
starting culture volumes can be used to generate the same amount of
plasmid as larger non-CAPped or non-induced ones. Thus, smaller amounts
of the S1, S2, and S3 lysis buffers will be required.
Note
Use of the rules described above may deplete the S series of lysis
buffers supplied with the Nucleobond AX kits. Please refer to the
Properties and Applications Guide for the formulas of these buffers when
they need to be prepared.
Nucleobond is a trademark of Macherey-Nagel, Germany.
Copywrite 1995 Dr. Kevin Doyle for The Nest Group, Inc.
The Nest Group, Inc. 45 Valley Road, Southborough, MA 1-800-347-6378
FAX# 508-485-5736 email: nes...@world.std.com
Email me if you need additional help. We are in the process of setting
up an FTP site but it will take a little time before we are ready.
Best regards,
Amos Heckendorf (nes...@world.std.com) 800-347-6378 ;508-481-6223
The Nest Group , Value Added Resellers for:
Macherey-Nagel,mfg of Nucleobond AX, Nucleotrap,NucleoSpin
PolyLC, mfg of HPLC packings for Biomolecule Separations
The Separations Group, mfg of Vydac
Mr. O.Coutelle (NIMR)
I have frequently used miniprep DNA for taq terminator cycle sequencing on the ABI, with no problems
whatsoever. I found that standard alkaline minipreps worked as well as Wizzard preps, and I get reads
in excess of 350bp. I use about 1 ug of miniprep DNA directly in the sequenceing reaction. I have
also got excellent results with quiagen midiprep cosmid DNA, which I use to walk on with custom
made walking primers. There was virtually no noise, and I could get up to 450bp.
It seems to me, that the problem you have lies somewhere else. You could also PCR amplify your
plasmid inserts, and directly sequence the PCR products after PEG ppt with a nested primer, this
always works for me.
Oliver Coutelle
email: ol...@med.cam.ac.uk
Zophonias O. Jonsson
> Does anyone out there know what the best way to prepare low-copy number
> plasmid (mini-prep scale) for ABI automated sequencing?
I am sure there will be many different opinions, but anyway here are my USD
0.05.
I had inserts in a ColE1 containing low copy number plasmid dyeDeoxy
sequenced and read with ABI sequencer. To get enough plasmid I grew the
cells in TB, no chloramphenicol tricks.
Around here most people agree that Quiagen's Quiawells are very suitable
for the ABI. They are easy and quick if you have many samples and give
very good sequences. The only problem is that they are _EXPENSIVE_ .
So I and a couple of friends decided to play a little bit. We isolated
plasmids with a variety of methods, sequenced and compaired the results.
The Quiagen products performed well as did carefully done phenol extracted
minipreps (treated with RNaseA). ordinary wizard gave just a few dozen
bases worth of useful sequence, but the most astonishing result was that
our CRASH minipreps worked beautifully.
That was a big surprise indeed, because the DNA is very dirty but somehow
it doesn't seem to matter. We have used such minipres for ordinary dsDNA
sequencing with good results as well. So here is how we do it if anyone
has nerve enough to try. Anyway, It's cheap and fast and you have all the
reagents at hand I am sure.
Basically it is Alkaline lysis as described in Sambrook (Maniatis) but
after the addition of Solution II (NaOH/SDS) HEAT THE PREP FOR 10 min AT 70
C. This probably denaturates your DNA irreversibly but it also breaks down
most of the RNA and denaturates proteins. After addition of solution III
spin down the precipitated proteins, SDS and other particulate junk and
transfer the sup. to a new tube. Then add 250 ul of Isopropanol to
precipitate the DNA, spin and wash once or twice with 70% EtOH. Resuspend
in 10 ul H2O. NO PHENOL, NO CHLOROFORM, NO KIT!
I hope this helps someone.
Zophonias
_______________________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie
Universitat Zurich-Irchel
Switzerland
Amos H Heckendorf
there know what the best way to prepare low-copy number plasmid :
(mini-prep scale) for ABI automated sequencing? We've tried Promega
Wizards : (Magic uesd to work but that's another story), but they don't
like to yield : any sequence even with additional precipitations/ washed
to remove excess salt. : We tried QIAGEN 20 tips-- they work great down
the hall where they use pUC : and cosmids, but the yields for pKK223-3 and
pGP1-2 derived vectors are : deplorable. Folk here have even put CsCl
banded DNA down a 20 tip to find : that they are only able to recover 10%
or so. What we've settled on is either doing larger minpreps (or rather a
D. Walentas : e-mail: c.d.wa...@bioc.cam.ac.uk
Mr Walentas:
I somehow failed to get this on your thread on my newsreader. Please
forgive me if this is duplicated on your end.
We have seen problems with low yields for large DNA isolation and have a
product (NucleobondAX) we support in North America which does quite well
on P1 constructs and DNA out to 300KB. Yields on an IPTG-induced one
liter P1 culture are typically greater than 300 ug when the reagents in
the kit are used according to the attached protocol.
Please respect the copyright
Copyright 1995 Dr. Kevin Doyle for The Nest Group, Inc.
The Nest Group, Inc. 45 Valley Road, Southborough, MA, USA 508-481-6223
FAX# 508-485-5736
email: nes...@world.std.com
Nucleobondª Technical Notes
Number 5.1.2-5
Special Conditions When Using Nucleobond AX to Purify P1 Constructs and
copyright 1995 Dr. Kevin Doyle for The Nest Group, Inc.
The Nest Group, Inc. 45 Valley Road, Southborough, MA 508-481-6223 FAX#
508-485-5736
email: nes...@world.std.com
Hope this helps with your special case of low copy number or large size
DNA isolation. Feel free to email me if you need any clarification on
techniques. If you want to get in touch with Macherey-Nagel in Europe, cal
49-2421-69827
Regards,
Amos Heckendorf (nes...@world.std.com) 800-347-6378 ; 508-481-6223
The Nest Group , Value Added Resellers for:
Macherey-Nagel, mfg of Nucleobond AX
PolyLC, mfg of HPLC packings for Biomolecule Separations
The Separations Group, mfg of Vydac
TosoHaas, mfg of TSK Gels