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sequencing of miniprep dna

David Micklem WCI drm21 at mole.bio.cam.ac.uk
Wed Jul 20 06:57:13 EST 1994


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...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)223 334129      Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)223 334089

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