Silver Staining Sequencing

oliver at bf.bio.bg.ac.yu oliver at bf.bio.bg.ac.yu
Sat Nov 22 20:51:43 EST 1997


In article <34759C73.61D0 at aki.ku.dk>,
  Jens Mollerup <jmollerup at aki.ku.dk> wrote:
>
> Hi Netters,
> In near future I will start sequencing some EST clones. I intend
> to use a non-radioactive sequencing method, however, I do not know
> if the silver staining protocol (from Promega - called SILVER
> SEQUENCE) will work properly. Has anyone experience with this method
> or some other non-radioactive sequencing method?
>
> Please let me know
>
> M. Sc. Jens Mollerup
> University of Copenhagen
> The August Krogh Institute

Hi Jens, I am doing a silver sequencing for a month or so, but I don't
have any silver staining kit. Instead, I use comercialy available
components (Ag nitrate, CaCO3, and so on). I obtained a fairly good
results. As stated in Promega protocol, U should use ultrapure (or double
destiled) water, and other highest quality chemicals. Also, if using
cycle-sequencing (as we do), you should try to obtain as much of the
sequenced product as possible (raise the concentration of the sequencing
primer and nucleotides as well as of the template, and work at the lowest
possible annealing temperature). I don't need to keep photographic record
of the obtained sequences, but glass plates with the gel on are stable
for months. Also, I am submerging the plate in the bind silan solution,
instead of applying it to the plate (the first approach is suggested by
the bind silane manufacturer, and the second by Promega protocol). If you
encounter any trouble with this protocol, please write me. Oliver
Stojkovic Faculty of Sciences, University of Belgrade, Center for
advancement and application of PCR oliver at bf.bio.bg.ac.yu

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