Double stranded DNA sequencing

Charles J. O'Kelly okellyc at megasun.BCH.UMontreal.CA
Sun Mar 27 19:29:03 EST 1994


In article <Stephen_Lasky-270394110423 at tonto-slip12.cis.brown.edu>, Stephen_Lasky at brown.edu (Stephen R. Lasky) writes:
|> 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.  

They're calling them "Wizard PCR preps" now ... and associated with the name change is a major
change in the composition of the resin.  Be careful.  We've found with ca. 600 bp PCR products
that the column "eats" about 200 ng of the loaded DNA.  If your prep has less DNA than this ...
(the "Magic" formula gave better yields in our hands).

Charley O'Kelly
Mad Protistologist, neophyte mol biologist
okellyc at bch.umontreal.ca

|> 
|> ************************************************************************
|> Stephen R. Lasky Ph.D.  Brown University/Roger Williams Medical Center
|> LandLine: 401-456-6572          	       	e-mail: Stephen_Lasky at brown.edu
|> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
|> The art of science is of vital importance to the state.  It is a matter of
|> life and death, a road to safety or to ruin.  Hence under no circumstances
|> can it be neglected.  SunTzu (paraphrased)
|> *************************************************************************
|> 
|> 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
|> >  



More information about the Methods mailing list