Lowcopy number Minipreps and Automated Seq

Amos H Heckendorf nestgrp at world.std.com
Mon Feb 6 10:14:36 EST 1995


Christopher Walentas (cdw1000 at bioc.cam.ac.uk) wrote: : 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 : e-mail: c.d.walentas at 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: nestgrp at 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.  
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: nestgrp at 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 (nestgrp at 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




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