Promega silver sequencing.

[NEAL MUSTO]

We have used silver sequencing for limited sequencing with reasonable
success. We have made just a few modifications to the protocol.
   1. As stated in the original DNA silver staining paper, we use only
PDA as a crosslinker. The background is significantly better than using
BIS.
   2. The water rinse after the Ag soak is just a rapid plunge into DW,
a quick drain and right into the developer.
   3. The developer temperature shopuld be close to 4C.
With these modifications we get respectable sequence information, though
I think not as good as with radioactivity. The Amount of sequence is not
as great.
One more thing, the use of the EDP film makes the reading of the gel a
bit easier since it exagerates the contrast of the bands.
IMHO silver seq is good for a lab that occasionaly needs to do limited
sequencing, such as checking a sequence after mutagenesis.

Neal Musto
Cell Biology
Georgetown University