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Hi all,
I'm doing my first RNAseq experiment. I followed the NuGen Ovation Ultralow Library V2 protocol which suggests throughout to elute after every bead purification step in TE (low EDTA). This is also what they suggest for the final elution of amplified library. Obviously, they don't see a problem with 0.1mM EDTA in the library (my reasoning is that it will inevitably be further diluted when pooling the libraries and won't pose such an issue?). However, I only read our genomics facility requirements now and I see that they do not want any EDTA in the libraries, to prevent the sequencing reaction failure. Do you think I should re-purify my libraries with beads (if so, in what ratio) and elute in the non-EDTA containing buffer? Molarities of my libraries are quite variable - from 6 nM up to 30 nM, so I am afraid of losing a lot if I re-purify.
Thank you!
I'm doing my first RNAseq experiment. I followed the NuGen Ovation Ultralow Library V2 protocol which suggests throughout to elute after every bead purification step in TE (low EDTA). This is also what they suggest for the final elution of amplified library. Obviously, they don't see a problem with 0.1mM EDTA in the library (my reasoning is that it will inevitably be further diluted when pooling the libraries and won't pose such an issue?). However, I only read our genomics facility requirements now and I see that they do not want any EDTA in the libraries, to prevent the sequencing reaction failure. Do you think I should re-purify my libraries with beads (if so, in what ratio) and elute in the non-EDTA containing buffer? Molarities of my libraries are quite variable - from 6 nM up to 30 nM, so I am afraid of losing a lot if I re-purify.
Thank you!
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