LARGE PLASMIDS

the End jgraham at bronze.ucs.indiana.edu
Fri Dec 11 10:10:11 EST 1992


Ian,

I've had much luck sith a 11.5 Kb low copy (pSC101) plasmid. I grow in TB
"terrific broth" as described in the new Maniatis (24g YE, 12g Tryp, glycerol,
PO4 buffer) with plenty of aeration (tube roller).

A standard alkaline lysis followed by neutralizing with a half volume of 7.5 M
ammonium acetate will do the job. When a 1.5 ml miniprep is resuspended in
50 ul, 5 ul will give a nice digest on an agorose gel.

One trick you should try is to freeze your resuspended cell pellet after
you have added lysozyme in the first step of your miniprep. This seems
to have a marginal beneficail effect with DH5 as a host, but may be
more important with other strains. I saw it in the inital report of 
pGB1, and it is a well know method of unleashing autolytic activity in
one of my favorite organisms, Staph. aureus.
J. Graham
Biology and Chemistry Departments
Indiana University -Bloomington
Jim
J. GrahaUniversity -Bloomington
...............It is necessary to be slightly underemployed if
.............one is to do something realy significant. " J. D. Watson..........



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