Boiling Method for Minipreps

Martin Kennedy mkennedy at chmeds.ac.nz
Thu Aug 5 17:29:03 EST 1993


In article <1993Aug4.221947.9602 at alw.nih.gov>, Jim Owens <jow at helix.nih.gov> writes:
> In article <9308032222.AA20703 at calshp.cals.wisc.edu> ,
> jchen at CALSHP.CALS.WISC.EDU writes:
>>As a matter of fact, I prefer HB101 as a
>>host because cells are easy to resuspend into the lysozyme buffer.
> 
> Somewhere in my collection of references there is a report about lysozyme
> not being necessary, and even reducing plasmid yield, in some strains of
> _E. coli_.  So I could not help wondering if anyone has tried a boiling
> miniprep without adding lysozyme.

> Good luck,
> 
> Jim Owens

Jim, 

We dropped lysozyme out of our boiling mini-prep procedure a year or two back. 
We always use Bluescript/DH5 alpha combinations, and it works fine.  There was
a suggestion that the yields were slightly down in the absence of lysozyme, but
we still get plenty of DNA (ca 10ug per 1.5ml O/N) and it sequences fine.  This
gives us a really minimal protocol, especially as we use a multiple dispensor
(Eppendorf with Combitip attachment) for the STET and ethanol steps.  I feel
the Bluescript yields are better than pUC, and certainly much better than
pBR322, so I can't vouch for not using lysozyme in all plasmid/host
combinations.  Incidentally, I seem to recall the original Holmes and Quigley
paper stating that the lysozyme was only present to help bring down the lysed
cell debris etc during the centrifugation.


-- 
Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
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