Primers designed for dsRNA synthesis:
- dsPer48F.1: TAATACGACTCACTATAGGGACCCCCTACCCCAACTATCC
- dsPer48R.1: TAATACGACTCACTATAGGGGTCGCAGAGCATCGTTTCC
Control primers (without T7 promoter): AMplicon size(205bp)
- Per48F.1: ACCCCCTACCCCAACTATCC
- Per48R.1: GTCGCAGAGCATCGTTTCC
At 60°C annealing temperature, control primers amplify successfully while dsRNA primers fail to produce a band." I have also tried different temperature ranging from 50°C-62°C but still id dint get expected results.
Any one can help me with this
Libor Krásný
If I understand it correctly, it is first DNA synthesis you want to achieve and what fails is PCR amplification with primers containing the T7 promoter sequence for subsequent RNA synthesis. If this is so, have you tried to use the 205 bp amplicon obtained with the control primers as the template for PCR with your specific primers?
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