The CDS 3, 8, 9, 10 and 11 were amplified using polymerase chain reaction (PCR) and the primers information summarized in Table 1 . The primers were designed to generate amplicons of 200–400 bp. Then HRM were performed using LightCycler® 96 Instrument (Roche Diagnostics, Roche Instrument Center AG, Rotkreuz, Switzerland). The amplifications test were performed in 10 μL volumes containing 10 ng of genomic DNA, 2 μmol/L primers, 2.5 mmol/L MgCl2 and 5 μL 2X LightCycler® 96 High Resolution Melting Master (Roche Diagnostics) buffer. PCR cycling included an initial preincubation at 95 °C for 10 min, followed by 45 cycles of 10s at 95 °C, from 65 °C to a “touchdown” at 55 °C, and 30s at 72 °C. The melting program included three steps: denaturation at 95 °C for 10s, renaturation at 65 °C for 1 min, and a subsequent melting cycle consists of a continuous fluorescent reading from 60 °C to 90 °C at a rate of 25 acquisitions per °C. The detected mutations were confirmed with an independent PCR and bidirectional sequencing.
Lightcycler 96 high resolution melting master
Manufactured by Roche
The 2X LightCycler® 96 High Resolution Melting Master is a laboratory equipment product designed for high-resolution melting analysis. It provides the necessary reagents for performing this analytical technique.
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Lab products found in correlation
2 protocols using lightcycler 96 high resolution melting master
1
HRM Analysis of CDS Variants
2
Genetic Profiling of Autism Spectrum Disorder
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The CDS 3, 8, 9, 10 and 11 were ampli ed using polymerase chain reaction (PCR) and the primers information summarized in Table 1. The primers were designed to generate amplicons of 200-400 bp. Then HRM were performed using LightCycler® 96 Instrument (Roche Diagnostics, Roche Instrument Center AG, Rotkreuz, Switzerland) in DNA samples from autism patients and healthy controls. The ampli cations were performed in 10 µL volumes containing 10 ng of genomic DNA, 2 µmol/L primers, 2.5 mmol/L MgCl2 and 5 µL 2X LightCycler® 96 High Resolution Melting Master (Roche Diagnostics) buffer. PCR cycling included an initial preincubation at 95℃ for 10 min, followed by 45 cycles of 10 s at 95 ℃, from 65℃ to a "touchdown" at 55℃, and 30 s at 72℃. The melting program included three steps: denaturation at 95℃ for 10 s, renaturation at 65℃ for 1 min, and a subsequent melting cycle consists of a continuous uorescent reading from 60℃ to 90℃ at a rate of 25 acquisitions per ℃. Mutations were con rmed with an independent PCR and bidirectional sequencing.
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