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fangw 01-17-2014 02:18 PM

Fail in ligation of 5' RNA adapter

I am looking for help with library preparation in MiSeq. I have tried to ligate 5' RNA adapter (33bp) to RNA (22bp) already ligated to 3' DNA adapter (33bp), but it did not show any band at about 88bp. I have tested that the 3' adapter ligation is fine. The concentration of 5' adapter is 5 uM, and my ligation condition is 1h at 20 degree using T4 RNA ligase. I also tried 37 degree and overnight ligation, did not work. This 5' adapter terminates with "U". Will it affect ligation process? Any idea how to solve this problem? Thanks.:)

Genohub 01-22-2014 08:45 PM

small rna ligation of 5'adapter
Sometimes it's difficult to visualize a doubly ligated small rna product (rna with a 3'ligated and 5'ligated adapter). The ligation efficiencies are pretty low, making it difficult to visualize on a gel. I would try running your sample on a bioanalyzer. You could also make a synthetic control RNA (with 5'phosphate and 3'OH) to check your 3 and 5'ligations. You'll be able to see this much better than small RNA isolated from cells or tissue.

- Genohub

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