I am doing a Megascript RNA precipitation reaction using Sp6 polymerase.
After the reaction, I add 1uL DNase for 15mins, then 1uL EDTA to quench.
After adjusting volume to 50uL, I add 5uL 5M Ammonium Acetate, followed by 3 volumes of EtOH. At this point, a cloudy white precipitate forms, which sometimes forms larger snowflakey aggregates.
I let sit at -20ªC for 30mins, then spin down, and wash with 70% EtOH. I then let the pellet (a very sizable, very visible and very white pellet) air dry for a few minutes before adding 50 uL water.
After the first resuspension, I repeat the protocol one more time, in theory to fully remove any dNTPs. Final working volume is 25uL.
At this point, I go to the nanodrop and measure RNA conc., which comes out to 5ug/uL, with a 260/280 of 1.6 and 260/230 of 1.9.
I ran two different types of gels. One is a non-denaturing 2% TBE gel and a 2-log ladder, and one is a non-denaturing 2% TBE gel but using a special RNA loading buffer that denatures my product, in theory and an RNA ladder (loaded using the same buffer as the sample). For all gels, I loaded 1uL of RNA solution.
2 things are immediately noticeable.
1) There are not 4ug/uL RNA in my solution -- this suggests a lot of dNTPs carried over.
2) Bands are present in the non-denaturing gel that are present in the denaturing gel, but I have trouble believing that secondary structure could cause RNA to travel 10x faster on a gel.
So my question is: What is happening?
A few things that concern me:
A large white pellet is usually salt and or protein. RNA pellets from something like a Trizol extraction are generally a bit white, but after 75% EtOH washes and air drying they tend to be mostly transparent if pure. This could be a sign of precipitating a lot of salts, perhaps NTPs or degraded RNA based on the nanodrop reading.
Precipitating NTPs seems likely, as I just read that the MEGAscript kit is made to utilise large amounts of NTPs. Perhaps you could run the RNA through a sephadex column to remove the NTPs before precipitating. This should help with the false nanodrop reading if it is the true cause, and would allow for buffer exchange for something more compatible with precipitation.
If this doesn't help, I would try omitting the DNase step, which introduces a new buffer and more enzymes as well as a warm incubation that could be degrading your RNA. Try skipping it and running your RNA on a gel to see if anything changes.
As far as the gels go...I don't understand why you want to run the RNA on a non-denaturing gel for analytical purposes and then compare them to your denaturing gel. I'm not sure what your concern is here.
Best of luck.
David, you need to follow the Megascript protocol. Not EtOH precipitation, but Phenol-Chloroform/Isopropyl Alcohol precipitation. Nucleotides, salts, and proteins are likely to co-precipitate with your RNA in your current protocol.
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