sequenase kit

Bill Melchior, NCTR/FDA WMELCHIOR at NTET.NCTR.FDA.GOV
Wed Jun 17 08:29:56 EST 1992


>trying to sequence ds DNA using the Sequenase kit (USB).
>Her 1.9 kb insert is contained in an M13 plasmid vector.  She is
>preparing the DNA using large scale plasmid preps (alkline lysis) and
>purifying plasmid DNA through CsCl gradients.  She has also tried
>alkline lysis mini-preps and plasmid recovery using silica.  Her problem
>is that all she gets on the sequencing gel is a large smear.

Since my sequencing is limited to pBR322, my comments are limited, but I have
had one thought:  Since the DNA is evidently clean (CsCl), and I assume the 
same is true for the primer, the problem sounds like it could be with the
gel.  Is the gel stuck to the plates firmly enough to prevent the sample from
running OUTSIDE the gel?  Has your colleague done any controls and gotten
good results (eg M13 w/o insert?)

(I'm a little confused about the reference to ds sequencing with M13.  I thought
M13 was generally used for SINGLE strand sequencing, though I know it CAN be
made double stranded.)



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