Hello all
I'm doing RNA seq, and using AMPure XP beads for cleanup steps and size selection of my RNA lib. Before using beads, my RNA concentration is good, however, after using Ampure beads the RNA concentration decreased too much. Most of my fragments had been washed away or were gone.
What should I do?
Before using Ampure beads, allow the beads to sit at room temp 30 minutes, and also, I used 80% ethanol for washing step.
I tried different volume ratio of beads to samples. From 1X tp 2X.
Thank you
Hello Elham Amini. E.A,
Can you specify the protocol you are following ?
It looks like you could have lost your fragments by discarding the beads or supernatant...
Juliette
Thank you dear Juliette for your reply.
Exactly, I have lost my fragments by discarding supernatant. Because, I read the RNA concentration before and after clean up step. They significantly decrease after washing and size selection using beads.
For protocol, I used the same manufacturer protocols. I put the ampure xp beads Beckman beads out of fridge at least 30 min to become warm before start the procedures. Based on the protocol I chose the beads ration. I need mRNA with size of 150bp and more. The ratio I used 1.6X and 1.8 X.
Thanks for your help