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:
Thank you so much for your time and help!
Best regards,
Check the pH values of your loading buffer and of the urea-PAAG after polymerization (you can just use a strip of pH paper and either drop the buffer on it or press it against the gel at the bottom or top). The pH must be near neutral. It may happen that tRNA is being degraded upon preparation of the loading sample or inside the gel because of alkaline hydrolysis. The loading buffer usually contains formamide, which may alkalinize the sample due to decomposition to ammonia and formate. Similarly, urea may decompose to ammonia and carbonate, which results in high pH in the gel. Since DNA is more resistant to alkaline hydrolysis, the ladder may survive the alkalinization just fine while tRNA is fully destroyed.
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