Please, I will do a tetra-primer ARMS-PCR experiment. I need any one who has experience in this method to advice me about the work way. I bought primers- master mix- template. I need the precautions and how I can face the problems that may face me during work
The precautions outlined in the paper are quite good.
Os it a already established protocol that you are following or have you designed your own assay?
If you have designed your own assay make sure that the expected size of the PCR products is above 100 bp so that you can visualize them on a gel and the difference between the two products for different alleles is at least 50 bp.
The optimization of the melting temperature is a crucial step and you might also want to optimize the DNA concentration to be used (by taking different DNA concentrations and running a PCR).
One of the most crucial steps is the MgCl2 concentration and you must do a gradient of MgCl2 as well. This will provide specificity to the products. In my experience something between 1mM to 3mM can be used.
A precaution while running the gels, don't use the buffer for running more than 3-4 gels for good resolution.
Hope this helps.
Good Luck!!
Here is the whole procedure : Ye, S., S. Dhillon, X. Ke et al. 2001. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res. 29(17):E88–E88. 392
It's what we use in the article :Human Biology |Issue 3 Volume 83 Article 4 6-1-2011 Lactase Persistence in Central Asia: Phenotype, Genotype, and Evolution
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