In Ule 's CLIP protocol, there was a 3' RNA linker, 5'-P-GUGUCAGUCACUUCCAGCGG-3'-puromycin. I can not understand why added a puromycin. what is the meaning of puromycin.
This is a resistance cassette but from your descriptio I don't get the reason and also no idea for what it is used? Do you want to do PAR-Clip? I don't see the point please give more information first
regards
Sven
In this case, it is not a puromycin "resistance cassette" actually, but a puromycin moiety added to the 3'end of the oligo used for ligating to RNA tags pulled down in CLIP experiments. This moiety is added to the 3' end so that the oligo can ONLY ligate to CLIP tags at its 5'end, creating 5'CLIPtag-LigationOligo-3'-Puro. This is what they used in older CLIP-seq protocols, but Jernej Ule's group now mostly uses the iCLIP method with a 3'pre-adenylated DNA ligation oligo. I have successfully used this technique and I recommend it. See
http://www.sciencedirect.com/science/article/pii/S1046202313004143
for a detailed protocol. Cheers
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques