Supermix for probes, Droplet digital PCR (ddPCR) assays, DG8TM cartridges and Droplet Generation Oil were obtained from Bio-Rad Laboratories Ltd., Hertfordshire, UK. Deionized water, sodium chloride, sodium hydroxide, phosphate-buffered saline (PBS), sodium nitrate, 4-aminobenzoic acid, hydrochloric acid, ethanolamine, 2-(N-morpholino) ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hexammineruthenium (III) chloride, potassium ferricyanide, potassium chloride and potassium ferrocyanide were all purchased from Sigma–Aldrich, (Dorset, UK). Two hundred and fifty units of HotStarTaq Plus and dNTP Mix, PCR Grade (200 μL), were purchased from Qiagen, (Manchester, UK). Phusion Direct PCR kit was purchased from thermo fisher scientific (Renfrew, UK).
Dg8tm cartridge
Manufactured by Bio-Rad
Sourced in United Kingdom
The DG8TM cartridges are a laboratory equipment product manufactured by Bio-Rad. They are designed for use in the analysis and processing of samples in various scientific applications. The core function of the DG8TM cartridges is to provide a standardized and efficient method for sample preparation and handling.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Merck Group (4 mentions)
Qx200 droplet reader, by Bio-Rad (4 mentions)
Qx200 droplet generator, by Bio-Rad (4 mentions)
Ddpcr supermix, by Bio-Rad (4 mentions)
Potassium ferrocyanide, by Merck Group (3 mentions)
4 aminobenzoic acid, by Merck Group (3 mentions)
Hotstartaq plus, by Qiagen (3 mentions)
Phusion direct pcr kit, by Thermo Fisher Scientific (3 mentions)
Supermix for probes, by Bio-Rad (3 mentions)
Potassium ferricyanide, by Merck Group (3 mentions)
Hydrochloric acid (hcl), by Merck Group (3 mentions)
Potassium chloride (kcl), by Merck Group (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
Sodium nitrate, by Merck Group (3 mentions)
Ethanolamine, by Merck Group (3 mentions)
N hydroxysuccinimide, by Merck Group (3 mentions)
Droplet generation oil, by Bio-Rad (3 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride, by Merck Group (2 mentions)
2 n morpholino ethanesulfonic acid mes, by Merck Group (2 mentions)
Deionized water, by Merck Group (2 mentions)
8 protocols using dg8tm cartridge
1
Droplet Digital PCR Assay Protocol
2
Quantification of TRPV2 expression using ddPCR
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About 10 ng of cDNA generated from 1000 ng of total RNA reverse transcribed with the transcriptor first-strand cDNA synthesis kit (Roche) was used. Droplet digital PCR (ddPCR) was performed using the QX200™ Droplet Digital™ PCR system (Bio-Rad Laboratories) including the droplet generator and the reader. Reactions were prepared in 20 µL volumes with QX200™ ddPCR™ EvaGreen Supermix (Bio-Rad Laboratories). As above, we used Hs_TRPV2_1_SG QuantiTect Primer Assay (Qiagen) and DEDD as a housekeeping gene. After droplet generation in DG8TM Cartridges (Bio-Rad Laboratories), ddPCR™ 96-well PCR plates containing reactions in duplex were loaded onto Veriti 96-well Thermal Cycler (Applied Biosystems) and cycled as follows: 5 min at 95°C, 40x (30 s at 95°C and 60 s at 58°C), 5 min at 4°C, 5 min at 90°C, and held at 4°C. QuantaSoft Software v1.7 (Bio-Rad Laboratories) was used to analyze the output of QX200™ Droplet Reader (Bio-Rad Laboratories). Relative TRPV2 expression was calculated as ratio of copies/µL as follows:
3
Validation of Genetic Variant Imputation
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Imputation results for rs2055272 were further validated by TaqMan genotyping (assay C_3025729_20, ThermoFisher Scientific) in 102 patients (98.03% concordance). Validation was performed by droplet digital PCR (ddPCR) approach using a QX200 Droplet Generator (BioRad) and the data was analyzed by QuantaSoft software (BioRad). Briefly, a ddPCR mastermix was prepared containing 11 μl 2× ddPCR Supermix (Bio-Rad), 1.1 μl 20× Taqman SNP Genotyping Assay (Applied Biosystems), and 7.9 μl nuclease-free water (Qiagen) per sample. The mastermix was prepared at room temperature and 20 μl was added to 2 μl (5 ng) of each DNA sample. Samples were loaded into individual wells of DG8TM cartridges (BioRad), and droplets were generated using a QX200 Droplet Generator (BioRad). For each sample, 40 μl of droplet mix was then transferred to a 96-well plate, and PCR was performed in a thermal cycler using the following cycling conditions: 95°C × 10 min; 40 cycles of [94°C × 30s, 60°C × 60s]; 98°C × 10s; 40°C × 10 min. The Bio-Rad QX200 Droplet Reader was then used to assess droplets as positive or negative based on fluorescence amplitude. The QuantaSoft software (BioRad) was used to analyze droplet data.
4
Confirming Mutations via ddPCR Analysis
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The mutations identified by WGS were further confirmed by Droplet Digital Polymerase Chain Reaction (ddPCR) technique using a QX200 Droplet Generator (BioRad) and the data was analyzed by QuantaSoft software (BioRad). Briefly, a ddPCR mastermix was prepared containing 11 μl 2X ddPCR Supermix (BioRad), 1.1 μl 20X TaqMan SNP Genotyping Assay (BioRad, ThermoFisher Scientific; Supplementary Table 6 ), and 7.9 μl nuclease-free water (Qiagen) per sample. The mastermix was prepared at room temperature and 20 μl was added to 2 μl (5 ng) of each DNA sample. Samples were loaded into individual wells of DG8TM cartridges (BioRad), and droplets were generated using a QX200 Droplet Generator (BioRad). For each sample, 40 μl of droplet mix was then transferred to a 96-well plate, and PCR was performed in a thermal cycler using the following cycling conditions: 95 °C × 10 min; 40 cycles of [94 °C × 30 s, 60 °C × 60 s]; 98 °C × 10 s; 40 C × 10 min. The BioRad QX200 Droplet Reader was then used to assess droplets as positive or negative based on fluorescence amplitude. The QuantaSoft software (BioRad) was used to analyze droplet data.
5
Droplet digital PCR protocol
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Droplet digital PCR (ddPCR) assays, Supermix for probes, DG8TM cartridges and Droplet Generation Oil were obtained from BioRad Laboratories Ltd., UK. Deionised water, sodium chloride, phosphate buffered saline (PBS), sodium nitrate, 4-aminobenzoic acid, hydrochloric acid, ethanolamine, 2-(N-morpholino) ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), potassium ferricyanide, potassium chloride and potassium ferrocyanide were all purchased from Sigma-Aldrich (Dorset, UK). 250 units HotStarTaq Plus and dNTP Mix, PCR Grade (200 μL) were purchased from Qiagen, (Manchester, UK). Phusion direct PCR kit was purchased from thermo scientific, (Renfrew, UK).
6
Digital PCR Assays for Quantification
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Droplet digital PCR (ddPCR) assays, Supermix for probes, DG8TM cartridges and Droplet Generation Oil were obtained from Bio-Rad Laboratories Ltd., Hertfordshire, UK. Deionized water, sodium chloride, phosphate-buffered saline (PBS), sodium nitrate, 4-aminobenzoic acid, hydrochloric acid, ethanolamine, 2-(N-morpholino) ethanesulfonic acid (MES), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), potassium ferricyanide, potassium chloride and potassium ferrocyanide were all purchased from Sigma-Aldrich, (Dorset, UK). Two hundred and fifty units of HotStarTaq Plus and dNTP Mix, PCR Grade (200 μL), were purchased from Qiagen, (Manchester, UK). Phusion Direct PCR kit was purchased from thermo fisher scientific, (Renfrew, UK).
7
Droplet Digital PCR Quantification Protocol
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Droplet digital PCR (ddPCR) assays were performed using the QX200 system (Bio-Rad, Hercules, CA, USA) in a 20 μL reaction mixture that included 10 μL of 2× ddPCR Supermix (Bio-Rad), 0.8 μL of each primer (10 μM), 0.4 μL of probe (10 μM), 1 μL of template DNA, and a final addition of ddH2O to 20 μL. A total of 20 μL of mixture together with 70 μL of DG oil was added into a Bio-rad DG8TM cartridge, which was placed into a QX200 Droplet Generator to generate water-in-oil droplets. 40 μL of droplets was transferred into the 96-well plate to perform PCR amplification on a C1000 Touch TM thermal cycler (Bio-Rad, Hercules, CA, USA) following this program: 95 °C for 10 min; 45 cycles of 94 °C for 30 s, (55–62 °C) for 1 min; 98 °C for 10 min; final cooling to 4 °C. After amplification, the 96-well plate was loaded onto a QX200 droplet reader (Bio-rad, Hercules, CA, USA) to read droplets using Bio-rad QuantaSoftTM software (v1.7.4.0917). The default setting for thresholds was initially adopted to allow the distinction between positive droplets and negative droplets. If the default setting failed to work, the threshold was manually set up to analyze droplets.
8
Droplet Digital PCR Analysis of Soybean Transcripts
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Total RNA was extracted from soybean seed slices using the RNeasy Plant Mini Kit (Qiagen) according to the supplier's instructions. cDNA was synthesized using the qScript cDNA SuperMix (Quantabio) from 1 µg total RNA previously treated with DNase I (Merck). For droplet generation, 20 µl of PCR reaction (cDNA, primers and the Bio-Rad ddPCR supermix) and 70 µl of droplet generation oil were transferred to the middle and to the bottom rows respectively of a DG8 TM Cartridge before insertion into a QX200 Droplet Generator. The genes used and the primer sequences are included in Supplementary Table S4. Droplets were transferred to a 96-well plate for PCR amplification in a thermal cycler C1000 Touch TM . The cycling protocol was 95 ºC enzyme activation for 5 min followed by 40 cycles of a two-step cycling protocol of 95 Cº for 30 seconds and Tm (Supplementary Table S4) for 1 min, then 4 ºC for 5 min and 90 ºC for 5min. Following PCR amplification, the plate containing the droplets was placed in a QX200 droplet reader. Droplet digital PCR (ddPCR) data was analysed with Bio-Rad QuantaSoft Analysis Pro Software. The Glycine max ATP synthase subunit 1 (Glyma12g02310) and SKIP16 (Glyma12g05510) were used as internal references (Hu et al., 2009) .
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