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sequencing of miniprep dna

Martin Kennedy mkennedy at chmeds.ac.nz
Fri Jul 15 00:18:43 EST 1994


In article <300bjd$hs0 at mserv1.dl.ac.uk>, m-brazil at nimr.mrc.ac.uk writes:
> Forwarded message:
>> From server-daemon at dl.ac.uk Wed Jul 13 05:36:57 1994
         ...scads of stuff deleted...
> 
> Dear Martin,
> Do you sequence directly from your minipreps without cleaning them up with
> phenol/chloroform? 
> 
> melanie 
> m-brazil at nimr.mrc.ac.uk
> 

Melanie - yes! These are the cruddiest, dirtiest preps ever, with big pellets, 
and they sequence beautifully.  Instead of going to great lengths adding more 
protein, various detergents etc back into minipreps to get the perfect ds seq 
template, just try the crude boil prep.  But remember to use DH5 alpha!!!  
Just harvest cells, resuspend in STET add lysozyme and boil for 90", spin 10 
minutes and tip into an equal vol of isoprop. Spin 5 minutes, rinse in 70%, 
resuspend in water (or TE) add RNase.  Use alkaline denaturation, and then 
sequence as normal.  48 preps in a morning is a doddle.

Warning: this DNA is no good for ABI or other automated sequencer 
technologies, but is ideal for Klenow/Sequenase type reactions. 
-- 
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
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
	      Phone (64-3)364-0880  Fax (64-3)364-0750



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