Hello, I'm trying to create a 12% polyacrylamide gel that can work in both nondenaturing and denaturing conditions, using 7M urea to observe the ssDNA, miRNA, and extended miRNA (polyadenylation solution). I'm using a 1* TBE buffer, and I also added an RNA ladder, but I haven't been able to visualise any bands. I've made numerous attempts to denature my sample, both at 95 degrees Celsius and without it. I also have tried to run the gel without urea and with higher concentrations of polyacrylamide. Nothing has worked. Has anyone successfully completed this protocol and could potentially assist me? Please advise me on the most effective method for visualising ssDNA, miRNA, and extended miRNA (polyadenylation solution), as well as the appropriate percentage for each.
What stain are you using to visualize nucleic acids? Do you include a dye in your loading sample, and can you see the dye migrate along the gel?
12% gels should be fine, either denaturing or not. What is your method of detection? Can you see your marker bands? If you are using ethidium you will need microgram amounts. I'm guessing either you are not loading enough or your method of detection is not very sensitive.
Ciaran Daly I used SYBR Green II to visualize the band of RNA yes, the dye migrate down into two sections
SYBR Green is used sor dsDNA. For miRNA, I use SYBR Gold. Note that in this case, the staining must be done post-run and visualize immediately (if not, the staining is lost). As for the quantity, 5microg should be enough (maybe more is too much). Good luck!
Eva Romaní Thank you for the answer. I used Sybr green II and immediately put gel on after the run. Because I perform polyadenylation, Sybr Green II is effective for visualizing miRNA with a poly(A) tail.
- Badges
- Science topic