Hello everyone!
Currently, I am genotyping DNA samples via forced RFLP-PCR method. However, most of the time, I get the second non-specific band. The main problem is that when I use this PCR product, the restriction enzyme does not work. From this, I understood that I need perfect PCR band. I assumed it's probably a problem with primer design but sometimes I could get clear band. Primers and PCR procedure were retrieved from previous studies (articles). I tried to increase an annealing temperature, and also ran gradient PCR. However, it does not work!
*Depending on your question about using a PCR product and the restriction enzyme (RE) doesn't work, If you targeting specific gene, maybe the sequence of this gene doesn't have the restriction site that specific for your current RE or the RE doesn't work, especially you already have bands in the attached figures.
*So, simply, can you change the RE?
There are many online tools to defined the restriction enzymes, such as; NEBcutter V2.0 (https://www.labtools.us/nebcutter-v2-0/) based on the previous sequence of the target gene. Worth mentioning, your problem not related to the primers or PCR product, it`s related to Restriction fragment length polymorphism (RFLP) technique.
* If it's not working, then you have to check the conditions of incubation (time, temperature,...etc.) for RFLP technique.
*Also, about the second band, I think it`s not specific band, may also, you need to reduce the volume of the primer.
Thank you. However, when I got those clear bands, the RE worked well, but when I use the PCR product with those extra bands, RE did not work. That’s why I assumed that may be it depends on my PCR product. As I mentioned, this gene, locus, primers, RE have all been previously studied.
The cause of the second band is primer dimerization. You can check the self complementarity score of your primers. Sometimes it can be solved by changing the PCR conditions, usually increasing the annealing temperature, but if it didn't work you can try different primers.
However, to my knowledge, it shouldn't affect your digestion. Try to optimize the digestion conditions. Common causes of incomplete digestion can be:
1. enzyme amounts (too much or too little enzyme can cause incomplete digestion)
2. too much substrate (DNA)
3. presence of contaminants in the DNA sample can inhibit the enzymes such as nucleases, salts, organic solvents (e.g., phenol, chloroform, or alcohol), and detergents.
4. reaction volume
5. short incubation time**
7. buffer composition (use a suitable buffer that gives the highest activity of your restriction enzymes)
8. nonoptimal temperature or pH
Also, some restriction enzymes require cofactors for full activity. For instance, Esp3I (BsmBI) requires DTT, while Eco57I (AcuI) needs S-adenosylmethionine.
Another point to mention, restriction enzymes are often supplied in 50% glycerol, so the final concentration of glycerol in any reaction should be less than 5% to minimize the possibility of star activity.
If you really think the second band is the cause of digestion failure, you can simply eliminate the second band using gel purification and cut the upper band only.
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