I have been a bit reluctant to reply as it's so long since I did this stuff, but have you tried EtBr and uv to visualise the bands? I used to stain DNA gels with crystal violet to avoid uv damage which was quite good but nowhere near as sensitive as EtBr and uv. There are reports that you can increase the sensitivity of the visible stains by using combinations of dyes, but I could never get anywhere near the published sensitivity when I tried quite hard to do the same thing myself, so EtBr staining is probably your best bet. Also, if the binding of the dye to RNA is weaker than DNA (the web says ~10 fold), when you run the gel, the nucleic acid goes in one direction and the dye front goes in the other, so with EtBr at least, it's good to have it in the running buffer, I think.
Best wishes, Jon Cooper. jon.b.coo...@protonmail.com Sent from Proton Mail Android -------- Original Message -------- On 23/12/2024 14:49, Jay Jani wrote: > Hello everyone, > I am currently facing challenges in visualizing tRNA on urea-PAGE, and I hope > the community can help with advice or a detailed protocol. > Here’s what I’ve done so far: > > - tRNA Source: In vitro transcribed tRNA (~80bp), purified using the Trizol > method. > - Purity and Concentration: RNA has a 260/280 ratio that’s satisfactory with > a concentration of ~400 µM. > - Gel Setup: Using 10% denaturing PAGE (7.5 M urea). > - Staining and Detection: SybrGold for staining, and visualization with a > G:Box GelDoc system. > > Challenges: > > - Despite testing various tRNA concentrations (10,000 ng/µL to 50 ng/µL) and > combinations (± MgCl₂, heating vs. non-heating), I consistently fail to > visualize clear tRNA bands. The DNA ladder (50–500 bp) appears fine. > - Once, I observed a faint band at 100 µM, but nothing more reproducible. > > Additional Notes: > > - Buffers and gel apparatus were treated with DEPC water. > - Enzymatic kinetic assays with the same tRNA showed functional results, > confirming its integrity and folding. > - In future work, I also aim to visualize tRNA-protein complexes. > > If anyone could share a detailed protocol or advice on troubleshooting, it > would be immensely helpful! I am particularly looking for: > > - Ways to improve tRNA detection on urea-PAGE. > - Suggestions for optimal staining and visualization methods. > - Specific tips for preparing RNA for electrophoresis. > > Thank you so much for your time and help! > Jay, > C/O, Dr. Anju Pappachan, > Protein Engineering and Structural Biology Group, > School of Life Sciences, > Central University Of Gujarat, > Sector 30, 382030, > Gandhinagar, Gujarat > > --------------------------------------------------------------- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
