High-resolution melting (HRM) was performed with the LightCycler® 480 real-time polymerase chain reaction (PCR) system and High Resolution Master Mix (Roche; Indianapolis, IN) in accordance with manufacturer instructions and our laboratory protocol (Xiao, et al. 2010 (link)). Melting curves and difference plots were analyzed using Gene Scanning Software. For samples with shifted melting curves, PCR products were cleaned using ExoSAP-IT® (United States Biochemical, Santa Clara, CA) and sequenced in the forward and reverse directions on an Applied Biosystems (Grand Island, NY) 3130XL Genetic Analyzer (Table S2 ).
High resolution master mix
Manufactured by Roche
Sourced in Switzerland
High-Resolution Master mix is a concentrated solution of reagents, including DNA polymerase, nucleotides, and buffers, designed for use in high-resolution DNA amplification applications. It is formulated to provide reliable and consistent results.
Automatically generated - may contain errors
7 protocols using high resolution master mix
1
High-Resolution Melting Analysis Protocol
2
Genetic Screening of ATP5MC Genes in Dystonia and HSP
3
Real-Time PCR with High-Resolution Melting
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In a 20 μl assay, 1X of High-Resolution Master mix (Roche Molecular Diagnostics, Switzerland), 300nΜ each of forward and reverse primers, and 2.5 mM of MgCl2 were added. As described in the previous study [18 (link)], 5 ng of template DNA was added, and the assay was made up with PCR-grade distilled water. The assay strip tubes were loaded onto the LightCycler480®. The assay was optimized based on previously described method [18 (link)] with minor modifications. The standardized thermal cycler settings include - an initial denaturation at 95 °C for 15 min followed by 50 cycles of denaturation at 95 °C for 10 s, annealing at 65 °C for 10 s and extension at 72 °C for 30 s, with initial 10 cycles of touchdown (1 °C /cycle). This is followed by final denaturation at 95 °C for 1 min and cooling at 4 °C for 2 min. The high-resolution melting was performed from 65 °C to 95 °C at a ramp rate of 0.02 °C/s with 25 fluorescence data acquisition points, followed by cooling to 4 °C for 30 s.
4
Quantitative Coral DNA Normalization
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In order to convert conventional HRM into a quantitative assay for pooled samples, equal amounts of each individual’s DNA must be added to the initial asymmetric PCR reaction. This DNA normalization step is particularly critical for pooling organisms that may have known or unknown assemblages, infections or symbioses as the relative amounts of target and contaminating DNA can vary among samples. To circumvent these problems, we used quantitative PCR (qPCR) to accurately measure the quantity of coral DNA in individual samples. Primers to amplify the SNP locus C23209S177 [27 ] were used in a conventional qPCR reaction performed under the following conditions using a LightCycler 480 machine (Roche): 5 ng holobiont DNA, 0.1 μM each forward and reverse primers, 2 mM MgCl2 and 1x High Resolution Master Mix (Roche) in a 15 μl volume. Reactions were heated to 95 °C for 10 minutes, then cycled 55 times as follows: 40 s at 95 °C, 40 s at 60 °C, 40 s at 72 °C, then cooled to 40 °C and held for 20s. Concentrations of target DNA were normalized based on differences in quantification cycle (Cq) by assuming that the fold-difference per each Cq unit difference was equal to 1/E, where E is the amplification factor per PCR cycle for the particular primer set [28 ]. The accuracy of this approach was verified by re-amplifying the adjusted DNA concentrations again with the same primer pair.
5
Real-time qPCR protocol for DNA quantification
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In a 20μl assay, 1X of High-Resolution Master mix (Roche Molecular Diagnostics, Switzerland), 300nΜ each of forward and reverse primers, and 2.5mM of MgCl 2 were added. As described in the previous study [18] , 5ng of template DNA was added, and the assay was made up with PCR-grade distilled water. The assay strip tubes were loaded onto the LightCycler480 ® . The assay was optimized based on previously described method [18] with minor modi cations. The standardized thermal cycler settings include -an initial denaturation at 95°C for 15 minutes followed by 50 cycles of denaturation at 95°C for 10 seconds, annealing at 65°C for 10 seconds and extension at 72°C for 30 seconds, with initial 10 cycles of touchdown (1°C /cycle). This is followed by nal denaturation at 95°C for 1 minute and cooling at 4°C for 2 minutes. The high-resolution melting was performed from 65°C to 95°C at a ramp rate of 0.02°C/s with 25 uorescence data acquisition points, followed by cooling to 4°C for 30 seconds.
6
Real-time qPCR protocol for DNA quantification
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In a 20μl assay, 1X of High-Resolution Master mix (Roche Molecular Diagnostics, Switzerland), 300nΜ each of forward and reverse primers, and 2.5mM of MgCl 2 were added. As described in the previous study [18] , 5ng of template DNA was added, and the assay was made up with PCR-grade distilled water. The assay strip tubes were loaded onto the LightCycler480 ® . The assay was optimized based on previously described method [18] with minor modi cations. The standardized thermal cycler settings include -an initial denaturation at 95°C for 15 minutes followed by 50 cycles of denaturation at 95°C for 10 seconds, annealing at 65°C for 10 seconds and extension at 72°C for 30 seconds, with initial 10 cycles of touchdown (1°C /cycle). This is followed by nal denaturation at 95°C for 1 minute and cooling at 4°C for 2 minutes. The high-resolution melting was performed from 65°C to 95°C at a ramp rate of 0.02°C/s with 25 uorescence data acquisition points, followed by cooling to 4°C for 30 seconds.
7
High-Resolution Melt Curve Analysis
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In a 20μl assay, 1X of High-Resolution Master mix (Roche Molecular Diagnostics, Switzerland), 300nΜ each of forward and reverse primers, and 2.5mM of MgCl 2 were added. As described in the previous study [18] , 5ng of template DNA was added, and the assay was made up with PCR-grade distilled water. The assay strip-tubes were loaded onto the LightCycler480 ® . The assay was optimized based on previously described method [18] with minor modi cations. The standardized thermal cycler settings include -an initial denaturation at 95°C for 15 minutes followed by 50 cycles of denaturation at 95°C for 10 seconds, annealing at 65°C for 10 seconds and extension at 72°C for 30 seconds, with initial 10 cycles of touchdown (1°C /cycle). This is followed by nal denaturation at 95°C for 1 minute and cooling at 4°C for 2 minutes. The high-resolution melting was performed from 65°C to 95°C at a ramp rate of 0.02°C/s with 25 uorescence data acquisition points, followed by cooling to 4°C for 30 seconds.
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