Double stranded DNA sequencing

[gc]

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