plasmid prep with G+ bacteria

Steven Goldberg goldberg at bms.com
Tue Nov 29 10:17:15 EST 1994


> Does anyone out there know how to purify plasmids from Gram + bacteria--
> i want purify a plasmid from drug resistant Staph. sp. to see if the plasmid is 
> responsible for imparting resistance.  The Promega miniprep kit doesn't work.  
> i also tried to treat the cells with Lysostaphin for 15 min. and then did a 
> wizard miniprep.  it didn't work either.  i always see a smear on the gel.
> anyinput on this sue would be of great help.
> Thanks.
> Kannan Thiru, University of Kansas.

The major problem with DNA isolation from gram + organisms is making
sure you have good protoplasts before lysis (or you get a lot of de-
graded DNA).  I use the E. coli alkaline lysis procedure but first 
resuspend the cell pellet in 5-10 mg/ml lysozyme in 0.3 M sucrose/
25 mM EDTA/25 mM Tris-HCl pH 8.0 and incubate at 37oC for at least 
30 min.  Then I take a 100 ul sample and add 0.5 ul of 20% SDS to see
if the culture clears.  If not, keep incubating for another 30 min.  
When you can easily lyse the small sample, proceed with lysis of the entire sample, followed by KAcetate precipitation, etc.  You may have
to mix different lytic enzymes to achieve disruption of the cell wall.

  



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