SNP markers significantly associated with carotenoid cleavage in petals of ray florets were validated using the ASP-PCR analysis using 10 ng of genomic DNA of the parents and F1 individuals. Specific primers corresponding to SNPs were designed so that their 3′ terminal nucleotides perfectly corresponded with the SNP match with one allele (the specific allele) but had a 3′ mismatch with other alleles (the non-specific alleles) (see Supplementary Table S2 ). ASP-PCR were performed using each primer pair under conditions of 95 °C for 30 s, 30 cycles of 95 °C for 5 s and 60 °C for 30 s, with a SYBR Premix Ex Taq II Tli RNase H plus (TaKaRa Bio, Shiga, Japan) on a thermal Cycler Dice Real-Time system (TaKaRa Bio, Shiga, Japan). Each SNP showed threshold cycle number response at 24–28 cycles.
Cycler dice real time system
Manufactured by Takara Bio
Sourced in Japan
The Cycler Dice Real-Time system is a laboratory equipment designed for the amplification and detection of DNA sequences in real-time. It is capable of performing quantitative polymerase chain reaction (qPCR) experiments.
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Lab products found in correlation
2 protocols using cycler dice real time system
1
SNP Marker Validation for Carotenoid Cleavage
2
SNP-based PCR Markers for P. horiana Resistance
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We investigated SNP-distinguishable PCR-based markers for the SNPs associated with P. horiana resistance. Allele-specific primers were designed corresponding to the SNPs (Supplemental Table 1 ) by browsing sequence reads using IGV software (Robinson et al. 2011 (link)). The SNP was validated by PCR using 8 ng of genomic DNA from the parents and F1 individuals. Touchdown PCR was performed with the following conditions: 95°C for 50 s, 40 cycles of 95°C for 5 s, annealing for 15 s, and 72°C for 20 s, where the annealing temperature is gradually reduced 2°C every third cycle from the initial annealing temperature of 66°C to the final annealing temperature 56°C. PCR was performed using the TB Green Premix Ex Taq II Tli RNase H plus kit (TaKaRa Bio, Shiga, Japan) on a thermal Cycler Dice Real-Time system (TaKaRa Bio).
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