Tail of the RNA band

[DNAartist]

Hi guys,

I am a chemistry graduate student. Currently I am transcripting a 50 bp long RNA from 70 bp long DNA. I am using the Ampliscribe T7 flash transcription Kits.
When I check the purity of purified RNA ( purified by cutting the 10% denaturing PAGE gel in TBE buffer) in 20% denaturing PAGE gel. I always got a significant tail of the RNA band ( which is in the lower mobility direction).

I am pretty sure that I have cutted the purification gel correctly. There should be no other impurities (such as template DNA, which has been digested by RNase free DNase I).


Could you please tell me what might be the reason and how can I get clean RNA product?

Thanks

[admin]

Hello there DNA Artist,
there could be several reasons.

THe first is that you somehow have a longer DNA. Either by not cutting fully or by PCR mistakes (it is longer than 70bp).

The other reason is that you have degradation, meaning that you get a smear instead of a clear band.

To get a clean RNA product, I would make sure you cut the DNA 100% to completion to have runoff transcripts and also use a sephadex column to get rid of nucleotides and small unfinished (but partly transcribed) RNAs.

I used RNAse free-Sephadex G-25 for less than 200bp RNAs (Roche carries them).

please let me know how you are doing.

cheers