Two droplet digital PCR (ddPCR) assays that targeted the exogenous rHSA gene and rice endogenous reference gene SPS were developed to evaluate the copy number of rHSA in GM rice line 114-7-2. The copy number of rHSA was calculated based on the ratio of rHSA and SPS (copy/copy). The primers and TaqMan probes for rHSA gene and SPS gene are listed in Supplementary Table S2. The ddPCR reaction was performed in a final volume of 20 µL and consisted of 10 µL of 2× ddPCR Supermix (Bio-Rad, USA), 1 µL 10 µM forward primer, 1 µL 10 µM reverse primer, 0.5 µL 10 µM probes, 1 µL DNA template, and 6.5 µL RNase-free and DNase-free water. The ddPCR was performed on a QX200 Droplet PCR platform (Bio-Rad, USA). The amplification conditions were: 5 min at 95 °C, 40 cycles of 30 s denaturation at 95 °C and 60 s extension at 60 °C, and a 10 min additional step at 72 °C. Droplet fluorescent signals were read on a QX200 Droplet Reader (Bio-Rad, USA). For each sample, the ddPCR reactions were repeated three times with triplicate samples.
Qx200 droplet pcr platform
Manufactured by Bio-Rad
Sourced in United States
The QX200 Droplet PCR platform is a digital PCR system designed for precise and sensitive quantification of nucleic acids. It utilizes a water-oil emulsion droplet technology to partition samples into thousands of individual reaction volumes, enabling the detection and quantification of rare target sequences.
Automatically generated - may contain errors
2 protocols using qx200 droplet pcr platform
1
Quantifying rHSA in GM Rice via ddPCR
2
Droplet Digital PCR Quantification Protocol
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The ddPCR was performed on a QX200 Droplet PCR platform (Bio-Rad, Pleasanton, CA, USA). ddPCR reaction was prepared with a final volume of 20 µL, which was comprised of 10 µL of 2× ddPCR Supermix (Bio-Rad, Pleasanton, CA, USA), 1 µL forward primer (10 μM), 1 µL reverse primer (10 μM), 0.4 µL probe (10 μM), 1 µL DNA template, and 6.6 µL ddH2O. After the 20 μL reaction mix was prepared, the mixture was transferred to 8-well cartridges for emulsion droplets generation using a QX200 droplet generator (Bio-Rad, Pleasanton, CA, USA). Then, the generated water-in-oil droplets were transferred to a 96-well plate for amplification with a T100 PCR cycler (Bio-Rad, Pleasanton, CA, USA). The program for ddPCR amplification was: 95 °C for 5 min; 40 cycles of 30 s at 95 °C and 60 s at 58 °C. After thermal cycling, 98 °C for 10 min and then cooled to 4 °C. After PCR amplification, the 96-well plate was transferred to a QX200 droplet reader (Bio-Rad, Pleasanton, CA, USA) for fluorescent signal monitoring. The readout data was analyzed using QuantaSoft (Bio-Rad, Pleasanton, CA, USA).
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