I need to genotype an association panel using simple sequence repeat (SSR) marker loci. As expected number of alleles in the panel cannot be predicted unlike in the case of segregating populations derived from bi-parental crosses; kindly suggest to me as to which is the better method for resolving the size-based SSR alleles? Metaphor agarose gel electrophoresis OR Polyacrylamide gel electrophoresis (PAGE)?
Hi
I would suggest you to use the Agarose electrophoresis and if possible send your samples for sequencing (or otherwise you can use fragment analysis) but only if you have fluorescent dye.
This might help you to better understand your results and to be generally more precise.
Please have a look at the following link:
https://www.researchgate.net/post/I-am-ordering-primers-for-SSR-markers-but-I-am-not-sure-on-what-basis-I-should-choose-the-fluorescent-dyes
Best regards
Hi Francesco Desiderio! Thanks for answering.
What agarose should I use: normal or metaphor agarose? I am not tagging my primers with fluorescent dyes.
Dear Kumaraswamy, in case you still need some sharing.
In my lab., We prefer to use SDS PAGE to resolve the allele diversity for the SSR panels. We use an old version of manual sequencing gel and stain the gel with silver nitrate staining system.
We have tried the agarose system, but sometimes are unable to obtain the expected resolution and the allele diversity is also under estimated compare to using the SDS PAGE.
SDS PAGE work well under our conditions. Depending on the pcr products, we usually are able to resolve most of the alleles for the evaluated ssr loci.
Some notes to be considered:
1) run preliminary sizing for the size ranges of pcr products of all ssr primer pairs,
2) for each gel plate, we run products of two primer pairs having enough size differences and lay the sample in such a way as well 1 is sample 1 with primer 1 and well 2 is sample 1 with primer 2, and so on. This sample lay out is to prevent possible leak from one column to the other of the same sample-primer combination. We usually run 50 samples (25+25) and two DNA markers per plate,
3) Make sure that the samples are loaded as single strand dna, after boiling up to 94 C, to obtain clear allele patterns. Complex allele patterns are probably because of the presence of a mixture of double strand and single strand molecules among the pcr product evaluated,
4) if your samples are more than 25 individuals per primer and you need to run more than one plate per primers, always take some samples from previous plates and use them as internal allele sizing for the additional plates. It will help to resolve allele identity among different plates for the same ssr primers.
Finally, all of the suggestions actually come around to your lab support. Do your lab have only horizontal agarose gel, or manual sequencing for SDS PAGE, or automatic sequencer ABI PRISM. Moreover, each approach you decide to use, will cost you differently. Therefore, you should use what is available in your lab and appropriate with the available budget.
In our case, SDS PAGE and manual sequencing apparatus plus Silver staining suit us well and fit with our budget. We have routinely used it to resolve many SSR works in our lab.
Good luck.
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