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sequencing of miniprep dna

m-brazil at nimr.mrc.ac.uk m-brazil at nimr.mrc.ac.uk
Wed Jul 13 04:19:41 EST 1994


Forwarded message:
> From server-daemon at dl.ac.uk Wed Jul 13 05:36:57 1994
> Resent-From: server-daemon <server-daemon at dl.ac.uk>
> Date: 13 Jul 94 15:21:18 +1200
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> Message-Id: <947130020.~INN-BLAa00158.bionet-news at dl.ac.uk>
> Resent-Message-Id: <1994Jul13.152118.554 at chmeds.ac.nz>
> Precedence: list
> From: mkennedy at chmeds.ac.nz (Martin Kennedy)
> Reply-To: mkennedy at chmeds.ac.nz (Martin Kennedy)
> Original-Sender: "bionet.molbio.methds-reagnts mail newsgroup" <server-daemon at uk.ac.dl>
> To: "bionet.molbio.methds-reagnts mail newsgroup" <bionet-news at uk.ac.dl>
> Subject: Re: Mini-Prep Blues
> Comments: List problems/queries to <biosci at daresbury.ac.uk>
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> X-Article-Number: bionet.molbio.methds-reagnts Msg # 7811
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> 
> In article <genecutl-080794200752 at kos2mac23.berkeley.edu>, genecutl at mendel.berkeley.edu (gc) writes:
> > 
> > 
> > I've been using a boiling-lysis protocol for mini-preps and it used
> > to work beautifully.  The protocol involves vortexing the cells in
> > STET buffer, boiling, spinning down the precipitated crap, then
> > EtOH ppt the sup.
> ........
> > The problem is now the mini-prep gods no longer smile on me and I'm
> > lucky to get a single sample to work out of twenty.  What can have gone
> > wrong?  Please don't tell me I have to go back to phenol/chloroform
> > hell.
> > 
> 
> Have you changed host strain, or scrounged someone elses competent cells?  
> Boiling lysis works beautifully, and plasmids sequence well, provided you use 
> an endonuclease-1 mutant like DH5 alpha. Any other strains give you DNA that 
> can degrade, and is not sequencable.  This isn't folklore - take it from me, 
> it's FACT!!!
>  
> Cheers,
> 
> Martin

Dear Martin,
Do you sequence directly from your minipreps without cleaning them up with
phenol/chloroform? 

melanie 
m-brazil at nimr.mrc.ac.uk







> 
> 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
> NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
> 	      Phone (64-3)364-0880  Fax (64-3)364-0750
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