I recently ran a gel electrophoresis experiment, but I encountered issues with my DNA ladder not separating properly and my bands appearing wavy and distorted. Here are the details of my setup:
- Voltage & Time: 80V for 45 minutes
- Gel Concentration: 1% agarose
- Buffer: 1X TBE (reused up to 3 times)
- Ladder: 1kb DNA ladder
- Expected Band Size: ~1.5kb
Despite these settings, my bands are not aligned in a straight line. Additionally, the DNA ladder does not show proper separation. What could be causing these issues, and how can I troubleshoot them?
I would appreciate any suggestions or insights. Thank you!
In agarose gel electrophoresis, these kinds of smiley bands occur when the buffer is compromised. The buffer can be reused in the tank for several times, but did you use 1X buffer while preparing the agarose gel? Is it too old buffer? It seems like the buffer composition is agarose gel is not accurate.
The gel may have been run too quickly. Recommended voltage for agarose gels is 5V/cm where the cm distance is the distance between the electrodedes in the gel tank. Try dropping the voltage so running the gel slower. Also make sure that the gel tank is horizontal on the bench. If one side of the gel is covered by more buffer than the other side then samples may run at different speeds (quicker on the shallow side of the gel)
Dear Hazwa,
To address this issue, I recommend following this protocol:
1. Use fresh TBE buffer for gel preparation and electrophoresis.
2. Set the voltage to 110 V, the current to 700 mA, and run the process for 60 minutes.
3. use the recommended concentration of the ladder as indicated in the product brochure.
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