To detect microsatellites using primers labeled in applied biosystems platform. I will use multiplexing (3 dyes) same specie. I need to make it as cheap as possible, in sequencing equipment (serie)
You have to purify the labeled primers to sequence microsatellite with a fragment analyzer.
Hello Miguel,
it is highly recommended to use purified (HPLC) primers to perform genotyping of SSRs with the application of fluorescently labeled primers. Fortunately, you need only small amounts of each primer for PCR (ca. 0.10 μM of each primer per sample in the reaction volume of 10μl) so it is quite cost-efficient. Moreover, only one primer from the certain pair needs labelling. I believe that only good quality reagents provide users with reliable and reproducible results.
You can decrease the costs of SSR genotyping by application of post-PCR multiplexing which, depend on the applied ABI platform, allows you to pool at least 3-4 sets of PCR products labelled by different dyes. If there is clear difference in the range of expected sizes of alleles for two SSR loci (no overlapping) you can try to use one dye for labelling both primer pairs and pool the obtained PCR products before fragment analysis. This approach can allow you to increase the number of pooled samples for one run of capillary electrophoresis, however, this method needs optimization.
Good luck.
Piotr
Yes, HPLC purification is recommended for labelled primers in multiplexing of microsatellite loci.
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