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RT PCR of viral RNA segment - (Sep/17/2008 )

Hallo, Ineed to amplify entire influenza virus RNA gene segment 2,3 kb in size. I had isolated whole RNA from alantoic fluid, lung and MDCK cells and convert to cDNA by RT with hexamers and specific primers. After PCR I gott wrong size band around 500bp. I tried several modification 52 degree of C for RT, prolong extension step in subsequent PCR I think, RNA template is not compet
has anyone suggestion for me
thanks

-baxapoptoaia-

QUOTE (baxapoptoaia @ Sep 17 2008, 03:30 AM)
Hallo, Ineed to amplify entire influenza virus RNA gene segment 2,3 kb in size. I had isolated whole RNA from alantoic fluid, lung and MDCK cells and convert to cDNA by RT with hexamers and specific primers. After PCR I gott wrong size band around 500bp. I tried several modification 52 degree of C for RT, prolong extension step in subsequent PCR I think, RNA template is not compet
has anyone suggestion for me
thanks


I would suggest you to do denaturation of your RNA at 65C for 15-20min and follow it by snap cooling on ice before proceeding to RT. This will help to remove any kind of secondary structures in RNA. Gud Luk

-paapu-