One of my friend is facing a problem with silver staining of sequencing gels in which the DNA sample bands along with the bands of marker are getting disappeared just after the developing solution is poured. This phenomenon is very strange in which we get a good staining as we put the developing solution and within few seconds the bands start becoming faint and finally no bends are seen whereas the gel turns slightly milky with brownish tinge instead of yellow. The developing solution contains Na2CO3 and formaldehyde. The staining protocol is as follows
Impregnating solution (AgNO3 + Ethanol + HNO3) -- 10min.
Rinse with D/W -- 5min.
Developing solution -- 2-5min. until the gel turns yellow
The gel is 6% polyacrylamide gel. We have tried various cross-linkings from 1.2% to 5% but there is no change in the result. The gel is prepared using 0.4mm spacers. I would also like to add that we have ruled out many of the possibilities like improper washing of glasswares, quality of milli-Q water -- by replacing the cartridge and even reagents. Other colleagues are getting the results with same protocol however their gel is not the sequencing one so the length is short and thickness is 0.8mm. Can anybody help us in explaining this sudden disappearence of a good quality staining within few seconds?