Hi everyone,
I need a solution for an issue that I have with my AFLP protocol.
Currently, I've been trying to make some AFLP's from a grass species that I study for my research project. However, I'm having a bit of an issue with the protocol.
The main problem that I see is there is a lot of big size DNA in the selective amplification step. I'm using a slightly modified protocol of Vos et al. (1995). MseI and EcoR I are the restriction enzymes used in the protocol with previously described adapters and primers. Upon screening in the gel, restriction digestion and pre-selective amplification seem to have worked and the DNA fragments are in the appropriate size range. However, after the selective amplification, I can only see nothing but bigger size DNA in the agarose gel. Upon recommendations by several experts, I changed the dilution in all steps, used a different taq polymerase, unified and separated restriction and ligation steps but I still get the same result. I have attached two gel images for your reference. Please take a look at them as well. Adding more to the disappointment, the protocol worked quite well when I did a test run to see if the protocol worked with my species. That was the only time it worked.
I cannot seem to narrow down where the problem is. If anyone out there has had the same problem or know how to fix this, your suggestions will be highly appreciated.
dear Piyal Karunarathne, I think, the number of nucleotides of your selective primers is rare.
so I suggested increase number of selective nucleotides.
Hi! By the way which kit are you using! Believe enzyme is intact.
Check the following links may answer your doubts.
Selective amplification problem in AFLP - Molecular Biology ...
http://www.protocol-online.org/biology-forums/posts/12709.html
Amplified Fragment Length Polymorphism (AFLP) - BioTechniques
http://www.biotechniques.com/BiotechniquesJournal/2010/May/DNA-and-General-PCR-Methods-Amplified-Fragment-Length-Polymorphism-AFLP/biotechniques-273562.html
Dear Mostafa Haghpanah, thanks a lot for the answer but I don't get what you exactly suggest here. Increasing the number of nucleotides in the primers will make them even rarer. Am I right? Or do you mean the number of nucleotides is not enough and to increase it?
Dear Jetty Ramadevi, thanks a lot for your answer. I used Invisorb plant mini kit for DNA extraction but I don't use any commercial kits for the AFLP protocol. I have come across these two threads/websites and I even met with one person who has written in the first thread. Apparently, she couldn't fix the problem.
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