I need to recover in vitro transcribed mRNAs from agarose gel, the size of mRNA is more than 1kb. I tried to cut the band and treat it with hot phenol. the protocol works fine but I don't have enough material at the end. Does anyone have suggestions?
Why do you need to extract it form the agarose gel? There may be a way around that. This extraction sounds a little cumbersome, if you can tell me what your goal is and why you are trying to extract it form the gel, perhaps I can come up with an easier solution.
I need to extract the mRNAs from the gel to use them for in vitro translation experiment through PURExpress™ In Vitro Protein Synthesis Kit from NEB.
Maria, we use this extraction kit for the same purpose.. works pretty well..
http://www.zymoresearch.com/rna/rna-clean-up/rna-gel-extraction/zymoclean-gel-rna-recovery-kit
For Sayantan... Do you have a good recovery? I need a preatty high concentration...
If your contaminats are represented by low molecular weight RNA molecules you might try ti dialyse the reaction mix (or use a gel filtration column) and then precipitate the dyalised/eluate. In general, my experience is that gel extraction is avery effective technique but has very low yields. It should be used only when you want to separate molecules that are very similar in terms of size or base composition
You certainly don't need agarose gel. You can separate your mRNA after in vitro transcription from template DNA and proteins using LiCl precipitation. The details are in this reference: Pokrovskaya, I.D., and Gurevich, V.V. In vitro transcription: preparative yields in analytical scale reactions. Anal. Biochem. 220, 420-423 (1994).
You can find more info in the original paper reference in the one above.
Hi Maria
I usually use electroelution for gel purifying RNAs. I can recommend GebaFlex tubes which are somewhat expensive, but usually gives at least 75% yield. If you add a dye to your agatose gel, you can actually visualize the RNA as it exits the gel and enters solution.
Alternatively, you can try the freeze'n'squeeze method (http://www.bio-rad.com/en-us/product/freeze-n-squeeze-dna-gel-extraction-spin-columns). The tubes are not sold as RNA grade, but in my experience they work just fine.
Jonas Wietek can you please share the modified protocol if you donot mind. i do the same, use mMachine mMassage SP6/T7 kits and clean and inject into frog oocytes and check expression and functionality by TEVC. but i am having problems with the mRNA yields. tried various purificatin methods but in vain. e tried LiCl too but it didnt yeild much. we need atleast 1ng/ul to be able to inject into frog oocytes. could you please help me.
Thanks,
Madhavi K
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