I have done RT-PCR using input RNA concentration of 400ng. Then I use 2uL of cDNA in 20uL reaction volume. I obtained Ct in range of 22-23. But now I am getting late Ct for a different sample with same input concentration of RNA. Only difference is now I am doing it in 10uL total volume of RT PCR. Kindly check the attached images.
Amy Klocko
Well, the amplification and melt curves look pretty good. I see you are now doing half-volume samples. Is your machine calibrated for that 10 ul well volume? I would suggest going back up to the 20 ul volumes and see if that helps. Keep in mind that smaller volume means you need more cycles to reach a detectable amount of amplification.
0 votes
0 thanks
More Sabahat Fatima's questions See All
Similar questions and discussions