Does anyone know what is the best/cheapest way of getting a fragment of RNA, up to 60 nucleotides long? In-vitro transription might be OK but I don't really know if it will give enough RNA for the crystallization trials. Buying such a long RNA oligo can be quite expensive, so I would appreciate any opinions or suggestions if you have experience in the subject.
According to NEB you can make up to 9 mg of a 300 mer RNA with their kit ($205), will you need to purify it? I would contact them about the potential yield for a 60 mer RNA, https://www.neb.com/products/e2040-t7-high-yield-rna-synthesis-kit
If you are not geared up to make your own T7 RNA Pol and buffers, I would recommend Ambion (now Life Sciences) MEGAshortscript.
http://www.lifetechnologies.com/order/catalog/product/AM1354
You need your sequence under a T7 promoter and I successfully do it from a PCR fragment with the upstream primer carrying a T7 sequence. Or even two long partly overlapping half-templates filled in. No cloning.
This kit has a very high polymerase concentration since this is the rate limiting step for short RNAs. For a 60 nt fragment processivity is not an issue.
Good luck!
Thanks for the answers! I'm going to have a look on both kits! The one from NEB seems to be cheaper but the truth is that for higher yields of my 60mer product it can be better to use the one from Ambion. The purification shouldn't be a problem since I want to bind the RNA to my protein so I could probably run SE chromatography once the complex is formed.
- Badges
- Science method
- Similar topics
- Methodology
- Crystallography