sequencing of miniprep dna

Viraj Master vam2 at midway.uchicago.edu
Mon Jul 25 17:50:26 EST 1994


Hi David,

If one uses the Holmes and Quigley STET recipe, how much salt do you add?


Thanks, 

Viraj


In article <30j3ep$25u at lyra.csx.cam.ac.uk>, drm21 at mole.bio.cam.ac.uk (David
Micklem (WCI)) wrote:

> In article <1994Jul20.134156.559 at chmeds.ac.nz> mkennedy at chmeds.ac.nz (Martin Kennedy) writes:
> <stuff deleted>
> >uses only one tube per prep.  Oh, and don't bother adding any more salt - 
> >there is enough in the STET, and enough RNA to cause all the nucleic acid to 
> >crash out.  Don't freeze the isoprop/DNA mix either, as this is likely to 
> >cause more crud to crash out - just spin immediately.
> >
> >Have fun!
> It's worth noting that there are several alternative recipes for STET, not all
> of which contain enough salt (eg the one I use).  The two recipes I know are:
> 
> Maniatis (oops Sambrook) et al:  0.1M NaCl
>                                  10mM Tris.Cl(pH8.0)
>                                  1mM EDTA (pH8.0)
>                                  5% Triton X-100
> 
> Holmes and Quigley (modified protocol from LMB Cambridge):
> 
>                                  8% sucrose
>                                  5% Triton X-100
>                                  50mM Tris.Cl pH8.0
>                                  50mM EDTA
> 
> I use the latter but most people I know use Maniatis'.  I add a mix of 350ul
> STET + 30ul lysozyme (5mg/ml in 50% glycerol).
> 
> Just my 2p worth...
> 
> David
> 
> _____________________________________________________________
> D.R.Micklem,
> Wellcome/CRC Institute,       Time flies like an arrow...
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> Tel: [+44] (0)223 334129      Email:drm21 at mole.bio.cam.ac.uk
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> 
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> i

-- 
Viraj Master
Dept. of Organismal Biology and Anatomy
University of Chicago



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