Double stranded DNA sequencing

Stephen R. Lasky Stephen_Lasky at brown.edu
Sun Mar 27 11:06:26 EST 1994


Try using Promega's Magic Mini-prep columns (or whatever they are calling
them now).  We get 100% sequencable DNA with these.  Fast, easy and give
readable sequences.  

************************************************************************
Stephen R. Lasky Ph.D.  Brown University/Roger Williams Medical Center
LandLine: 401-456-6572          	       	e-mail: Stephen_Lasky at brown.edu
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The art of science is of vital importance to the state.  It is a matter of
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In article <genecutl-160394165436 at kos2mac22.berkeley.edu>,
genecutl at mendel.berkeley.edu (gc) wrote:

> In article <1994Mar16.211722.674 at riscsm.scripps.edu>, Susan L Forsburg
> <susan_forsburg at qm.salk.edu> wrote:
> 
> > 
> > >RICHARD P. PHIPPS (PHIP at BPHVAX.BIOPHYSICS.ROCHESTER.EDU) wrote:
> > >: I am currently attempting to sequence double stranded plasmid DNA
> > from
> > >: minipreps using the USB sequenase 2.0 Kit.  Unfortunatly after
> > trying several
> > >: DNA isolation methods that I could think of or find I am not
> > having good
> > >: results in that > 70% of my reactions produce either no bands or
> > bands in all
> > >: four lanes all the way up the gel.  I am interested in a miniprep
> > DNA isolation
> > >: technique (Kit or otherwise) that reliably yields 50% or greater
> > readable
> > >: sequencing reactions.
> > >
> > >: Bob Burns
> 
> 
> I had the same kind of trouble in the past when i tried to 
> sequence alkaline lysis mini-preps with sequenase.  i 
> switched over to a boiling prep protocol and have had 
> virtually 100% sequencable mini-preps.  the protocol i use
> involves pelleting 1.5 ml of overnight DH5a, resuspending 
> in 250ul of STET, heating to 100 C for 1 minute 35 seconds 
> in a heating block,  adding RNase, spinning the tubes in 
> a microcentrifuge for 15 minutes, scooping out the pellet, 
> adding .5ml ethanol, spinning for 10 minutes, speedvacing, 
> and resuspending in 20ul TE.  These pellets have a whole lot
> of protein in them, but the look beautiful for digests, no
> degradation or anything.  If I plan on sequencing these,
> I add 250ul of TE and spin for 5 min to pellet all the
> insoluble crap and then add salt and re-precipitate the
> supernatant. I then use that  DNA directly in my dsDNA
> sequencing protocol.  As I've said, despite the large
> amount of crap that comes down in the initial precipitation,
> this protocol has given me consistently good quality
> sequences.
> 
> -- 
> --gc
>  



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