I am trying to extract RNA from bacteriophage MS2 filtrate of 10 log pfu/ml (propagated in E. coli C-3000 host) using Trizol and Qiamp viral RNA extraction kit. The RNA concentration was around 100-300ng/ul. I took 5ul of RNA for cDNA conversion in two steps using Primescript First strand cDNA synthesis kit (Takara) according to the manufacturer's instructions. I used MS2 2717F - 3031R (Dreier et al., 2005) primers and primers from Pecson et al., 2009 for PCR amplification of 1-5ul of cDNA. However, I am not getting any amplification and no bands are observed in the gel image. Please help me in this regard
Bowya Thangamuthu
What is the reaction volume of your RT-PCR? The amount of cDNA used should be less 1/10 of the total PCR reaction volume. I would suggest to dilute the cDNA and to repeat the RT-PCR with less template.
Thank you for your response. The reaction volume is 10ul. Even in diluted cDNA, I am getting negative results. Cp value is around 30-35.
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