RNA preparation from in vitro transcription - (Oct/03/2005 )
I use Roche in vitro transcription kit to make the rna from my pSFV vector. After the preparation and purification, I saw the rna pellet. The spectrum also told me high rna concentration (2 ug/ul) A260/A280 is around 2.1. The rna size should be 11kb. however, there is nothing i can find in my agarose gel. I load 4 ug (my rna marker is very clear).
Does someone here know what happened?
maybe it has to do with the kind and percentage of ag0rose gel to be used. i am not sure if a 11kb transcript would run stably on a low percentage agarose gel. Did u use TBE buffer.
Why do u need such a big transcript.
It's very difficult to transcribe such a big RNA. You got a high conc. of RNA, but most of them may be the prematured terminated transcripts. BTW, are u using circular DNA template or linearized template?
Run a formaldehyde denaturing gel to see this RNA. There is going to be all kinds of secondary structure.
I had this same problem! I found that I still had free NTPs after the reaction (even though they should've been removed during my purification steps). This was giving me false spec readings and, subsequently, I'd load way less RNA on my test gel than expected. Thus, no bands.
How did you purify it? I solved this problem by spinning it through a sephadex column.
11kb RNA is used because of pSFV vector
now, I found i can run in 0.5% TBE buffer agarose gel
the photometer is not believeable.
And I think i need to use some kit to remove the NTPs
Try a LiCL precipitation to remove the residual NTPs