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Gina_P 03-28-2013 10:09 AM

Beads sticking to tube during Ribo-Zero protocol
Hi all. I noticed that the Ribo-Zero magnetic beads tend to stick to the sides of the tube after you add your RNA sample and mix with pipetting and vortexing, and that this effect carries on through the end. When I finally pipette the 90 uL of sample from the magnetic stand, I usually just am very careful to guide the tip to the liquid without touching any sides since there will be beads stuck to it. However, recently I did the protocol and the sides of the tubes looked much cleaner than normal. My only thoughts are that I used a different brand of PCR tube during the incubation with the removal solution... any other ideas for why this may have changed? Or what your experiences are using Ribo-Zero?

ribodoc 05-06-2013 03:58 PM

We recently used ribo-zero for metabacteria and have >50% of our reads mapping to bacillus subtilis rRNA and e. coli rRNA. It appears that the capture failed for some reason. Does anyone perform QC to check for probe leakage? Anyone else experienced probe leakage? Its pretty frustrating.

Gina_P 05-07-2013 05:58 AM

We carried the libraries through despite the odd appearance of the beads which seems to be not unusual after all, and got less than 99.5% rRNA contamination with Oryza sativa using the plant leaf kit. I can't speak for the other kits and your application though. It would be worth it to contact Epicentre. They are very good at helping you troubleshoot until the protocol is working for you (providing some reagents for your trouble in the process).

ribodoc 05-07-2013 10:47 AM

Thanks for the reply. We've been using the metabacteria kit for awhile with good results so our recent experience is an outlier however our level of confidence in the product has decreased. Beyond basics such as making sure the beads are used and stored under the recommended conditions, Epicentre tech support was unable to help us. We have the contaminating sequences so we'll probably use PCR to QC the ribo-zero treated RNA for the capture oligos.

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