I am currently try to in vitro synthetize a small RNA. My DNA template is composed by two oligos that annealed for 20 bases, one of the two contain a T7 promoter. After annealing step (done in a termocycler with a temperature escalation program from 95 degrees to 25 degrees) I have elongated them with T4 polymerase (NEB) to obtain the DNA template (79bp). After I have checked the DNA template in a 2% agarose gel that have always confirmed the size of the DNA template. After I proceeded with the in vitro transcription using Ambion megashortscript kit. Subsequently i have checked the in vitro product on a 8M Urea polyacrylamide gel but i have obtained two bands: one at the right hight (62bases) and one higher (79bases). Supposing that the second higher band is not the DNA template, what could it be?
No other bands? Just those two?
A lot of RNA polymerases (especially those used in in vitro transcription kits) tend to add additional bases at the 3' end.
https://academic.oup.com/nar/article/46/18/9253/5096072
It's not typically a problem for most applications because most users are looking at large templates with included polyA sequence and additional weirdness at the end is thus irrelevant, but for short RNA it can be quite problematic (at one stage I was working with 7mers, 9mers and 18mers, with a T7 that generated those and...pretty much all variations in between and beyond).
Since you're running them on a gel anyway, you can just cut the band you want out and extract from it (that's what I ended up doing).
No other bands? Just those two?
A lot of RNA polymerases (especially those used in in vitro transcription kits) tend to add additional bases at the 3' end.
https://academic.oup.com/nar/article/46/18/9253/5096072
It's not typically a problem for most applications because most users are looking at large templates with included polyA sequence and additional weirdness at the end is thus irrelevant, but for short RNA it can be quite problematic (at one stage I was working with 7mers, 9mers and 18mers, with a T7 that generated those and...pretty much all variations in between and beyond).
Since you're running them on a gel anyway, you can just cut the band you want out and extract from it (that's what I ended up doing).
It is a good question.
As John Hildyard mentioned, I always observed longer transcript along with my desired transcript on Urea gel. The longer transcript was less intense than the desired one, and I always extract RNA from this desired band which worked fine.
We have a new manuscript that describes an approach to limit/prevent such additions. See Article Efficient inhibition of RNA self-primed extension by additio...
Also, be careful with gels. The darkest band may not be the one you want...
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