Hi all,
We're doing a Ribodepletion using the Illumina Ribo-Zero Plus rRNA Depletion Kit but did not realise we had to buy RNA clean up beads (Duh). Has anyone used AMpure XP beads for RNA before that could comment on yield/purity etc? OR if it's a big no no!
AMPure beads can be used for RNA. However, size selection likely is not the same for RNA and double-stranded DNA. I use AMPure beads for ssDNA and the cut-off volumes are quite different compared to dsDNA.
Miiko Sokka, I just saw your post. I am having troubles with size selection of ssDNA samples from dsDNA adapters. We have both Ampure XP and SPRIselect beads and optimization is taking quite some time. I would appreciate if you could share the volumes you are using (or share a publication mentioning it).
Thank you in advance for your help.
D.
Hi Diane Delobel, my goal was to size select ~500–3,000nt long ssDNA for which I optimized double size selection using AMPure beads. For double size selection, I first bound >3,000nt ssDNA ("large" in accompanied figure) using 0.50x beads, saved the supernatant (discarded the beads now containing >3,000nt ssDNA), then added beads to the supernatant for final 0.85x (which binds >500nt ssDNA) and proceeded as normally for AMPure purification. I added figure and notes from one experiment where you can find more details (I used ssRNA ladder for the optimization experiments; both RNA/ssDNA behave similarly with AMPure). The ssDNA (and RNA) behaves very differently from dsDNA, because ssDNA has a strong tendency to anneal and form double-stranded structures wherever it can. To minimize annealing, I used final 2M urea in the sample. One more thing, it is very difficult to bind less than 500nt ssDNA with AMPure beads, so it only works for larger fragments. Hope this helps.
One more thing, Diane Delobel. If you are working with ssDNA trying to make strand-specific libraries for sequencing, check out Article Genome-wide Nucleotide-Resolution Mapping of DNA Replication...
this publication. There you can find information on how to separate dsDNA adapters from the ssDNA (this clean-up step is critical for GLOE-seq!). Good luck!- Badges
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