Immidiate dissappearing of bands on DNA sequencing gels after silver staining

[himanshu]

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 geting dissappeared 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 insted 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 dissappearence of a good quality staining within few seconds?

[admin]

that is a weird problem Himanshu,
do you have any ideas on this or have solved it?

i will ask around

[kevin_a]

Himanshu,

I've seen this problem before but with a more complicated silver staining proceedure and I can't remember the root cause because it was so long ago.

With that in mind.....

I am thinking it was a simple matter of the gels not being completely rinsed with water between the steps. Since the silver ions are reduced/precipitate and get the color during development this indicated the ions are being oxidized from the acid remaining in the gel from your staining solution. It's also causing the bicarbonate to precipitate and give the milky white color.

As I recall the issue was with thick gels, too. Since the ethanol shrinks the gels, make sure you give the gels some good agitation during each step. Shorten the water wash to about 2-3 minutes, repeat several times with fresh water to make sure your gel completely swells back to orginal size.

Let me know if this helps or if am completely wrong.

K