I denature PCR product with formamide at 95 0C for 10 min. Then I run these denatured products on 14 % PAGE at 200 volts for 2 hrs. then I stained the gel with EtBr for 10 min. after destaining with distilled water, I got no bands for single stranded DNA while bands for Double stranded DNA are visible.
can anyone tell me weather formamide alone is enough for denaturation pcr product?
Does EtBr binds to single stranded DNA and stains it?
1) 70% formamide is enough
2) When single-stranded DNA form secondary structure (when it fold back upon themselves and base pair), then EtBr intercalate between base pairs.
Polyacrylamide quenches EtBr, reducing fluorescence. Also, unless the single stranded DNA forms a secondary structure (as Alisher mentions above), EtBr won't be as effective.
Is silver staining (AgNO3) not an option?
https://www.nationaldiagnostics.com/electrophoresis/article/ethidium-bromide-staining
as we are going to estimate polymorphism mean there must be some time of secondary structure formed by single stands after denaturation so?
Yes. Treatment with formamide is routine procedure before sequencing. Works fine.
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