Hi there.
Starting off by thanking you all for sharing your experiences in this forum, that's been really helpful!Sorry if this topic has been covered already but I could work out my problem so far reading other posts so there you go.
I'm having a bit of a problem in interpreting my Bioanalyzer trace on a cDNA library made with the Truseq mRNA kit. My RNA RIN was 9 so perfect I guess, then I've used 0.5,1 and 2 ug of RNA input (the figure shows just the result for the 0.5 ug input though), fragmentation time 8 minute which should give a fragment lenght of 300 bp (with adapters). All purification steps done with Ampure beads. In general I've followed the Illumina protocol religiously, no kiddin'. Then I quantify the library with either nanodrop (which gave me around 70 ng/uL for the three samples) and then with Qubit that gave me around 39-42 ng/uL. Never use nanodrop again!
I am quite surprise to see such a small pick compared to most of the other Bioanalyzer traces you all have shared so far. Also what surprise me most is too see such a wide range of fragment, resulting in a bump around 600/700 bp.
Have you had any experience of that at all? I've done a lot of work optimizing real time PCRs and I know how easily the amplification reaction can be inhibited by too much input, so I was wondering whether that's what is actually happening?
Any idea, suggestion, comment would be enormously appreciated!
Thank you all
Roberta
Starting off by thanking you all for sharing your experiences in this forum, that's been really helpful!Sorry if this topic has been covered already but I could work out my problem so far reading other posts so there you go.
I'm having a bit of a problem in interpreting my Bioanalyzer trace on a cDNA library made with the Truseq mRNA kit. My RNA RIN was 9 so perfect I guess, then I've used 0.5,1 and 2 ug of RNA input (the figure shows just the result for the 0.5 ug input though), fragmentation time 8 minute which should give a fragment lenght of 300 bp (with adapters). All purification steps done with Ampure beads. In general I've followed the Illumina protocol religiously, no kiddin'. Then I quantify the library with either nanodrop (which gave me around 70 ng/uL for the three samples) and then with Qubit that gave me around 39-42 ng/uL. Never use nanodrop again!
I am quite surprise to see such a small pick compared to most of the other Bioanalyzer traces you all have shared so far. Also what surprise me most is too see such a wide range of fragment, resulting in a bump around 600/700 bp.
Have you had any experience of that at all? I've done a lot of work optimizing real time PCRs and I know how easily the amplification reaction can be inhibited by too much input, so I was wondering whether that's what is actually happening?
Any idea, suggestion, comment would be enormously appreciated!
Thank you all
Roberta
Comment