Question

I'm researching a polymorphism using restriction fragment length polymorphism (RFLP) and gel electrophoresis.

After RFLP, I should see fragments at 141bp and 111bp, but I can not see in 2% agarose, what is your suggestion, dear friends?

Here is my gel recipe:

TBE: 0.5% 75ml
agarose: 1.5gr
Voltage: 65
Time: 1 hour
Unfortunately, I do not have the ability to change the methods.

Answer 1

The issue with not seeing the DNA fragments might be due to the high agarose concentration used in the gel. A lower percentage gel, optimal running time, and appropriate staining methods, like using Ethidium Bromide, could improve visualization of the 141bp and 111bp fragments expected from the RFLP.

Step-by-step explanation:

If you're unable to visualize the 141bp and 111bp fragments on a 2% agarose gel during RFLP analysis, the issue may lie in the resolution of the gel for small DNA fragments. A 2% agarose gel is quite dense, which is ideal for very small fragments, but if the DNA fragments are not well-separated, a lower concentration agarose gel might provide better resolution.

Generally, a 1-1.5% gel is sufficient for resolving fragments of 100bp to 1kb in size. Since you can't alter the method previously mentioned, you might want to ensure that the gel has fully set and that the running time and voltage are optimized for these small fragments. Additionally, make sure that the gel loading dyes and the staining method you're using are appropriate for the fragment sizes you're trying to visualize. EtBr (Ethidium Bromide) staining, for example, is commonly used and allows DNA visualization under UV light.