Tuesday, April 29th: Research Update, Technical
I'm transcribing a 65mer from an IDT 85mer with promoter and that part works great. Then I do a phenol/chloroform extraction and an EtOH precipitation. I've been trying to do precipitation with ammonium acetate to remove left over NTPs. But the RNA didn't show up after that ammonium acetate step (I did NaAc first). I'm worried it's degraded or the NH4Ac removed it, since it's small. The gel today should tell something. I ran this gel, labored over making complicated samples for the gel (had to make new ones and Nanodrop them, blah, blah) and also did another transcription reaction. But I'm storing my transcription reaction at -80C so I can keep precipitating it tomorrow. We shall SEE! I will get this to work eventually. I WILL. I'm less scared of phenol now. And better at EtOH precipitations.
College level layman translation
Future directions: From there, we will move to accomplishing this reaction on a gold nanoparticle (AuNP) in cells.
Research update translation: RNA transcription is the forming of an RNA strand from a DNA template using an RNA polymerase, in my case, T7. I purchased a template strand from Integrated DNA Technologies. They manufacturer DNA for researchers. One can only purchase a DNA fewer than 100 bp, which limits what I can do with purchased substrates.
After the transcription reaction, I add DNase I, an enzyme that cuts up the original DNA template, leaving only the RNA. I then do a phenol/chloroform extraction - adding an equal volume of phenol and chloroform to extract out the proteins (T7, DNase I), which denature and dissolve in the organic phenol layer, leaving the RNA in the aqueous phase.
I then precipitate the RNA with ethanol, first adding a salt. The salt neutralizes the charges in the backbone of the RNA and helps it precipitate. Different salts are used for different effects. Sodium acetate is the typical salt, while ammonium acetate is used to remove the left over NTPs from the polymerization reaction.
What this all means: I'm trying to get the above to work - i.e. proof that I have working RNA from all these procedures that I can use in my in vitro system and digest with DNAzymes. So far, that has not been determined. My last RNA gel after doing the ammonium acetate precipitation came up blank, meaning the RNA degraded or the ammonium acetate took away my RNA sample (it's technically only to be used for RNAs >100 bp).