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Hey there,
This is my first post here - thanks for reading it!
I'm rather lost. I'm prepping some samples for RNA-Seq, but in all four of my samples, I lose ~80-90% of my product at the mRNA fragmentation and cleanup step. I am using eukaryotic mRNA and am fragmenting with heat (94 C for 5 minutes) as per the protocol of the kit I'm using, which says these setting are ideal for fragmenting eukaryotic mRNA into 200 bp fragments.
After fragmentation, I am doing a clean-up of the mRNA using 1.8X AmPure XP beads. Somewhere during these two steps I am dropping from ~ 5 ng/ul mRNA to 0.8 ng/ul fragmented mRNA, on average as demonstrated by BioAnalyzer, with some of my samples dropping to dangerously low levels (.2 ng/ul). The average size of my fragments is ~ 100-150 bp.
Any idea why I'm losing so much product?
My goal for the fragment size is 200 bp and I'd like to retain as much of it as possible (obviously). I'm thinking my next step should be to decrease the amount of time at 94 C to 4 minutes and to increase the concentration of AmPure to 2.4X, but I would greatly appreciate any feedback.
This is my first post here - thanks for reading it!
I'm rather lost. I'm prepping some samples for RNA-Seq, but in all four of my samples, I lose ~80-90% of my product at the mRNA fragmentation and cleanup step. I am using eukaryotic mRNA and am fragmenting with heat (94 C for 5 minutes) as per the protocol of the kit I'm using, which says these setting are ideal for fragmenting eukaryotic mRNA into 200 bp fragments.
After fragmentation, I am doing a clean-up of the mRNA using 1.8X AmPure XP beads. Somewhere during these two steps I am dropping from ~ 5 ng/ul mRNA to 0.8 ng/ul fragmented mRNA, on average as demonstrated by BioAnalyzer, with some of my samples dropping to dangerously low levels (.2 ng/ul). The average size of my fragments is ~ 100-150 bp.
Any idea why I'm losing so much product?
My goal for the fragment size is 200 bp and I'd like to retain as much of it as possible (obviously). I'm thinking my next step should be to decrease the amount of time at 94 C to 4 minutes and to increase the concentration of AmPure to 2.4X, but I would greatly appreciate any feedback.
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