Boiling Method for Minipreps
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
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.
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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