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Hello,
My lab use's custom primer sets for HLA genotyping using the Illumina MiSEQ. We have optimized our multiplex primer sets for initial amplification using 96 well PCR plates. These reactions produce clean, cookie cutter bands when ran on 1.5% TBE gels. We have recently been experimenting with 384well plates and have found that when using the same reagent concentration, rxn volume (20ul) and thermal cycling conditions, the gels from the 384well rxns have non-specific bands and bad primer-dimers. I have tried lowering rxn volume to 10ul while maintaining original reagent proportions, varying mg2+ concentration, and increasing extension time all with unconvincing results. The 384well peltier blocks are calibrated and well maintained so it is confusing to me why we cant just scale up our procedure. Any one have some experience with switching to 384 plates? Some advice would be greatly appreciated. Thanks!
My lab use's custom primer sets for HLA genotyping using the Illumina MiSEQ. We have optimized our multiplex primer sets for initial amplification using 96 well PCR plates. These reactions produce clean, cookie cutter bands when ran on 1.5% TBE gels. We have recently been experimenting with 384well plates and have found that when using the same reagent concentration, rxn volume (20ul) and thermal cycling conditions, the gels from the 384well rxns have non-specific bands and bad primer-dimers. I have tried lowering rxn volume to 10ul while maintaining original reagent proportions, varying mg2+ concentration, and increasing extension time all with unconvincing results. The 384well peltier blocks are calibrated and well maintained so it is confusing to me why we cant just scale up our procedure. Any one have some experience with switching to 384 plates? Some advice would be greatly appreciated. Thanks!