Silver staining of DNA

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himanshu
himanshu's picture
Silver staining of DNA

Hi
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?

Thank you

Jason King
Jason King's picture
If the bands that are

If the bands that are disappearing are the shorter ones, it could be diffusion, especially if it happens in the 0.4mm gels but not in the 0.8mm gels.

Is it possible to check the gels after the bands have vanished by staining them with EtBr. This would tell you whether the DNA molecules have diffused out or if they are still there but no longer associated with silver precipitate.