single peak on RT pcr 2 bands on gel

[dasaras]

I designed a robust intron spanning primer set for FGF21 and ran the Real time pcr. Got a clean melting curve on triplicate, single peak but gel shows 2 distinct bands. Changed conditions, blasted the primers everything looks fine. Ran reaction again and same issue. Called tech support at Stratagene, and they do not seem to have any ideas except that the gel is contaminated!!! I ran other products on the gel and they have clean single sharp appropriate bands. Any thoughts will be appreciated.

[cds79]

Maybe the two peaks have same melting temperatures.... is the only thing i can think about.

I have had a strange experience with my gels. Recently we have changed the EtBr with a "safer" option called gelgreen. However, since then, for concentrated PCR products i see two bands where i have always got one. The second band appears just under the main one and it seems more like a shadow). Just in case you use the same dye, this may be another reason.

[cds79]

Maybe the two peaks have same melting temperatures.... is the only thing i can think about.

I have had a strange experience with my gels. Recently we have changed the EtBr with a "safer" option called gelgreen. However, since then, for concentrated PCR products i see two bands where i have always got one. The second band appears just under the main one and it seems more like a shadow). Just in case you use the same dye, this may be another reason.