LPS, P. multocida and restriction digests

MCGEED at HAL.HAHNEMANN.EDU MCGEED at HAL.HAHNEMANN.EDU
Wed Mar 8 13:04:01 EST 1995


On 8 Mar 1995 Kirsty.Townsend at net.bio.net wrote:

> Dear Fellow Micro-Netters,
> 
> I'm hoping someone out there can help me, because I'm having huge problems
> with restriction digests of particular strains of Pasteurella multocida.
> They are known to have a high LPS content in the cell wall, but I thought
> I was allowing for that during the million ether extractions. But the DNA
> I get at the end of it all, will cut with Eco RI but not with Pst I. I
> have tried using increased enzyme concentrations, different buffer etc
> etc but to no avail. Can somebody help me??? Other strains cut perfectly
> well with both enzymes, so it has to be the effect of the LPS. At present
> I do 3 phenol extractions, 3 with chloroform:isoamyl alcohol, and 3 with
> ether, precipitate with NaOAc and EtOH. Is there something else I should do?
> 
> Thanks in advance,
> 
> Kirsty Townsend
> Molecular Oncology
> QEII Medical Centre
> Nedlands  6009  WA
> AUSTRALIA
> ktownsen at eosin.path.uwa.edu.au
>  

  Kirsty, do you have any idea if the PstI site in the strain that's 
giving you trouble is methylated?  If it's methylated, then you won't be 
able to cut with your enzyme!!

David J. McGee
MCGEED at hal.hahnemann.edu

Graduate Student
McPHU (Medical College of Pennsylvania and Hahnemann University)
Center City Campus
15th and Vine Streets
Dept. Microbiology and Immunology M.S. 410
New College Building RM 10302
Philadelphia, PA 19102

Phone: 215-762-8275
Fax: 215-762-1004 

Happy 1995!  





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