SEQanswers - View Single Post - RNA-seq from single cells?

chaichontat · 12-22-2015, 09:28 AM

Hi Simone,

I'm trying to amplify one gene of interest; however, I couldn't get a specific band when I added the whole 10 ul of the RT reaction (final PCR volume was 25 ul). The band only appeared when I added 1 ul of the RT mix, suggesting that there's some inhibition of PCR by the RT mix.

However, there was one deviation from your protocol: I used the component version (not ReadyMix) of the KAPA HiFi Hot Start. Do you think that this could have been a factor?

Thanks!
Chaichontat

12-22-2015, 09:28 AM	#177
chaichontat Junior Member Location: Bangkok Join Date: Sep 2015 Posts: 3	Hi Simone, I'm trying to amplify one gene of interest; however, I couldn't get a specific band when I added the whole 10 ul of the RT reaction (final PCR volume was 25 ul). The band only appeared when I added 1 ul of the RT mix, suggesting that there's some inhibition of PCR by the RT mix. However, there was one deviation from your protocol: I used the component version (not ReadyMix) of the KAPA HiFi Hot Start. Do you think that this could have been a factor? Thanks! Chaichontat