Takara
SapphireAmp Fast PCR Master Mix
RR350A
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This reagent is designed to rapidly set up a hot start PCR. This made it ideal for large scale genotyping to assess a single nucleotide polymorphism of interest. The reagents were successful in a one-tube genotyping assay, and in generating PCR product for DNA sequencing.
PCR
Human genomic DNA
Genotyping was performed in a single tube assay (1) by allele-specific polymerase chain reaction (PCR) using primers containing a mismatch introduced at the second base from the 3’ end to improve allele specificity (2). As per the protocol, four oligonucleotide primers were included in the reaction mix (final volume of 20 ul). PCR parameters were an initial 60 sec of denaturation at 94o C, 5 sec of priming at 55o C, and 5 sec of extension at 72o C, for 30 cycles, PCR products were analyzed on a 1% agarose gel. References1) Waterfall CM, Cobb BD. Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis. Nucleic Acids Res, 2001;29:E119.2) Wong SH, Gochhait S, Malhotra D, Pettersson FH, Teo YY, Khor CC et al. Leprosy and the adaptation of human toll-like receptor 1. PLoS Pathog, 2010;6:e1000979.
The 2x master mix can be diluted four-fold, increasing the number of reactions and stretching research dollars.
The SapphireAmp Fast master mix gave excellent results with minimal optimization time. Assays results clearly delineate heterozygotes (three bands) from homozygotes (one band).
None
The mix includes a blue dye that obviates the time consuming need for mixing PCR products with tracking dyes.
Excellent product.
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