My friend and I have been doing SSCP using silver staining for the past few days.We run it at 110V, 350mA and for 3 hours.Her product size is 250bp. We are unable however to see lanes or the banding patterns. It was kept in fixing solution for 30 minutes and 20 minutes in developing solution. We used 5ml of 10% AgNO3. Does anyone have any suggestions regarding the same?
Edit : We modified the staining protocol a bit and new denaturation dye was prepared. We were able to get lanes. Thank you for the help.
How are you preparing your single stranded dna and how much are you running in each well. While you are troubleshooting this technique it may be useful to run a double stranded dna sample just to check that the silver staining is working
As an alternative to silver staining, did you try fluorescent dyes like Sybr Green II ? It is meant for staining RNA but it will also stain DNA, and although less sensitive with ss DNA than with ds DNA, it will still give a signal, and it is certainly less tedious than silver stain.
Are you fixing your sample in the gel.if not you may be washing the sample from the gel. I attach a good paper on silver staining for your interest which makes some important points concerning reagent preparation. Also check that you are running the gel in the right direction.
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