tRNA should be very easy to see. I suggest to buy cheap yeast tRNA as control and test different concentrations and different dyes until you can see it on the gel. I am not sure about SybrGold. It is a tricky dye and may take a long time to get in. Filter and excitation both need optimization. If you can't get your positive control to work, use other dyes. EtBr has no problem in visualizing tRNA at least in agarose gel.
On Mon, Dec 23, 2024 at 10:00 AM Jay Jani <jay.jani3...@gmail.com> 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: > > 1. Ways to improve tRNA detection on urea-PAGE. > 2. Suggestions for optimal staining and visualization methods. > 3. 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/
