Can I clarify a couple of points. You got a strong pcr band on agarose gel?

Did you get no stained band at all on PAGE or just the usual double stranded band but no single strand.

If you have no staining at all can you tell us how you did the silver staining?

Dear paul

we got strong bands on agarose, but only a gray gel after running on PAGE,

Thank you for your detailed information Mahmoud Khatib . It looks very good. I used to fix with methanol acetic acid but if you routinely use just acetic acid and it usually works then that is fine. Fixing is important to stop diffusion of the bands.

Another reason for failed sscp can be degradation or insufficient sample. Often the strongest band on an sscp gel is the double stranded re annealed dna and the single strand is weaker partly because there is less of it and partly because it is a smaller molecule so i would try a gel with increasing amounts of pcr product to see where you can confidently get a good image.

Another cause is degradation of the Amplimer. Formamide that has been stored for too long can degrade dna. Try using fresh formamide or deionising some old stuff if you have any monobed ion exchange resin in the lab. If not then find a newer bottle than the current one..

It may help diagnostics if you run another pcr product or a dna size ladder so that you can see that the staining part of the procedure is working properly

One additional point which will not help this problem but I used to find I got better polymorphic bands with 5% glycerol in the PAGE gel so that may be worth a try when you have a visible signal

Thank you Paul for sharing your experiences and complete guidance.o

Hi Mahmoud Khatib

thank you but I forgot 2 possibilities. The solutions containing formaldehyde should be made up fresh and not stored at 4c as this can inactivate formaldehyde.(the formaldehyde should be stored at room temperature)

Some times you can get a smear of ssDNA from sscp which makes it less visible. This is caused by the sample changing between 2 different conformations because both are stable at the gel running temperature. The solution to this is to either run at a lower voltage or in a cold to lower the temperature or to have a slight denaturant like glycerol in the gel.