MC1061/P3 minipreps

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Mon Dec 14 18:38:00 EST 1992


I realize that this reply is a bit late but our E mail port
was not working properly:  D.G.

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Greetings:

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.  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?

Thanks in advance,

David
haviland at kids.wustl.edu
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message------------------------------------------------------------------


Hi David,
    We have had this problem lately when we had to use another than our
favourite lab E.coli strain (which happens to be DH5alpha).  I think it was to
 generate
hypo methlyatedDNA in a dam- dcm- strain.  Anyway, when Mg++ was added to
 digest the plasmids we hadmajor amounts of degradation.  We solved it by 
being
 very careful the way we handle the bugs during the alkaline minis.  The two 
ml
 cultures were cooled in an ice water bath beforecentrifugation to pellet the 
cells
 (Which is done in the cold room). Everything was doneto keep the whole prep
 as cold as possible during extraction (normally with Dh5 alpha we
 never have to worry about temp).   I think another critical step is the
single phenol:chloroform extraction  after  the K acetate pptte.  
Normally with DH5 alpha we just vortex these for 5-10 sec and spin. 
 But we found if we vortexed the phenol:chloroform extractions from
 the other strain for 1-2 full minutes per tube the degradation problem is 
gone!
 We now do this with all strains we are unfamiliar with.  
Hope this helps.

Dan Gietz
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA



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