need protocol for precipitation of 13 mer mRNA - (May/18/2007 )
I am lookining for some info about what solutions do I need to precipitate a 13 mRNA.
What I am trying to do is to develop a capping efficiency assay. The mRNA was made in in-vitro transcription reaction so it is fairly clean. the experiment goes like this:
1)large mRNA (capped, 8kb) hybridized with a DNA probe(28 mer) about 14 bases from the capped end
2)treatment with RNase H (RNase H cuts the RNA hybridized to the DNA probe leaving a 13 mer capped and a ~8kb mRNA)
3)Precipitation of large RNA by standard protocols
4)Precipitation of small capped mRNA fragment by ?????
5)Test small capped mRNA by reverse phase (RP) chromatography
The idea is that the capped mRNA would be 14 mer long (13 mer+capped) while the uncapped mRNA would be only 13 mer. By measuring the ration I could determine the capping efficiency of the reaction. I have no problem resolving 1 nucleotide difference with my current RP protocol if I manage to solve the small mRNA fragment precipitation I think I had a chance to get this assay working
The other thing is that, if possible, I would like to stay away from kits.
Thanks in advance,
Add three vol of ethanol and 0.1 vol of 3M Na acetate pH 5.2 (or 0.1 vol NaCl 5M) and incubate overnight at -20. If the conc is low add a small amount of glycogen. I use 5-10 micrograms in a total vol of 2 ml. Spin at 14000 for 30 min at 4°C
why not use a size exclusion column profided by millipore,
YM30 should do the trick
Thanks for the info. I tried a 13 mer and it worked very well.