Hello!
I plan to run cDNA intended for RNA-seq in a gel and excise the band of interest in order to get rid of the adaptor from the library prep. Can anyone suggest me an optimized size exclusion protocol for small RNAs?
Thanks!
Dear Sir. Concerning your issue about Size exclusion optimized protocol for small RNA-seq. Size Exclusion Chromatography (SEC) can be carried out by equilibrate the column by gravity with 3 bed volumes of cold 1X Mammalian Polysome Lysis Buffer , Spin column 4 min @ 600 xg, then place the column in fresh collection tube, gently add 100 µl nuclease-treated sample to top of resin and spin 2 min @ 600 xg. Transfer flow-through to fresh tube on ice. Multiple columns per sample can be used if needed and samples pooled, then add 10 µl of 10% SDS to each eluted sample and extract RNA using Zymo RNA Clean & Concentrator™ kit. Elute RNA with 26 µl Nuclease free Water. Also a sucrose cushion can be used as an alternative to the MicroSpin S-400 columns to purify monosomes, then purify RNA from‘Total RNA’sample using Zymo RNA Clean & Concentrator kit (>17 nt protocol). Elute RNA with 26 µl Nuclease-Free Wate. Please refer to Appendix 8 for details. For more details see the following below links and attached file which may help you in your analysis:
http://www.epibio.com/docs/default-source/protocols/artseq-ribosome-profiling-kit-(mammalian).pdf?sfvrsn=16
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428589/
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA