Dear Experts,
I have some experience in population genetics studies in plant systems using various chloroplast intergenic spacers (sequence data). Now, I would like to analyse a very restricted plant population distributed in a "sky-Island' system using microsatellites as these markers provide high rate of polymorphism. As I have not used these markers before, I would like to know what are the limitations of my lab in terms of facilities. We have a normal gradient PCR and all other necessary equipments to perform standard DNA experiments. Is this enough to conduct experiments using microsatellites? and Can I use desalted primers for amplification for further visualization in ordinary gels?
Kindly help me by providing your valuable inputs.
Hello Rahul
It is possible but you need to have a PAGE system too to genotype your samples. Depends on the type of microsatellites and population variation, you may need a 20cm gel sandwich or a longer (30 cm) PAGE system. An optimized silver staining protocol is also crucial.
All the best
Hello! you will be able to perform PCRs using the desalted primers. Also, with the gradient thermal cycler you can find the optimum melting temperature for each primer pair and then explore to amplify several primers by essay. However, it is very difficult, or sometimes impossible to genotype in common agarose gel (since the aleles differ sometimes for a couple of basepairs. Alternatively, you may get the genotypes by using acrylamide gel (which is quite noxius and labour-intensive) or get one of the primers labeled by fluorescence, then perform the PCR just as done with the desalted primers and finally, send the PCR products (labeled) to analyze them in some lab that offers fragment analysis service (like Macrogen/Psomagen).
Hope it helps.
Regards
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