Promega Silver Sequencing

[bagley at FAUNA.UCDAVIS.EDU]

>
>Instead of the nice clear bands (I have seen a gel that worked so I believe it does work!), we get smears of DNA in the
>loaded lanes.  Even the markers smear, which maybe suggests it is a gel problem, but we have tested all our buffers etc
>extensively.  Towards the bottom of the gel we can often see faint, diffuse bands (with a little imagination - so important in
>science..), but nothing to get excited about.

Well I can tell you about some of the things I did to improve reading in our
lab.  However, all of these things were done during the learning curve and I
cannot guarantee that any are very important.
1. I changed the anneal and denature steps from 30s to 40s.  I felt the ramp
time on or thermocycler was quicker than what Promega used and this adjustment
seemed helpful.
2. Find the tubes with the lowest protein-binding properties you can find.  In
my case I moved from polycarbonate 96-well plates to 0.2 ml tubes.  I think
this improved the ladder a little, but it was not a big difference.
3. If background problems begin to accumulate, wash plates and staining trays
well with 3M HCl.  I do this after about every third gel.
4. I have dropped down to a 5% acrylamide gel, but am using the electrolyte
gradient technique (sodium acetate). I now regularly read 300 bases or more in
each lane (though I have to magnify it at the top).
5. Though Promega will deny it, I believe they have twice sent me bad tubes of
5x buffer.  The 5x buffer is just Tris and MgCl2 in low concentration, but two
times there was a precipitate that was difficult to get in solution.  When I
used these tubes of buffer I got poor, mostly unreadable gels.  If you saw a
precipitate in your 5x dump it and make your own.

Good luck,
Mark    (bagley at ansci.ucdavis.edu)