MC1061/P3 minipreps...

Basavaraju Shankarappa bsh at MED.PITT.EDU
Tue Dec 8 13:27:51 EST 1992


> Does anyone have any suggestions as to a good miniprep protocol for the MC1061/P3 cells?  They are not the most compliant cells when it comes to minipreps.
> Standard alkaline-minis provide degraded DNA.  
	In making DNA preps from most of the endonuclease rich strains like
yours and I believe HB101, I have had good results just by increasing the 
EDTA concentration to about 25 mM in the initial TE or GTE buffer which is
used to resuspend the bacterial pellet.  Although it is some work, I 
usually do the phenol-chloroform or at least chloroform extraction will
in most cases reduce the problems.

Promega mini-prep systems
> fail completely and yeild NO DNA.  An altered alkaline mini prep protocol
> (ala Maniatis) provides DNA but can not be digested.  Does anyone have any
> suggestions?

I would suggest that you can take the DNA obtained by this procedure and
subject it to promega mini-prep system.  Just a suggestion.

Raj Shankarappa
bsh at med.pitt.edu




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