The melting temperatures of the MhGgH variants were determined using a thermal shift (Thermofluor) assay. Each protein sample (0.5 mg ml−1 final concentration) was centrifuged at 13 000g and 4°C for 15 min, mixed with 5× SYPRO Orange (Life Technologies) in storage buffer and loaded into white 96-well PCR plates (Bio-Rad) sealed with Optical Quality Sealing Tape (Bio-Rad). The plate was heated from 25 to 95°C in 0.5°C steps with 30 s hold time per step on an iCycler iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad) and the fluorescence was followed using a Cy3 dye filter (545 nm excitation/585 nm emission). Each experiment was performed in triplicate. The melting curves were analysed using the CFX Manager software (Bio-Rad) and the melting temperature was determined as the inflection point of the melting curve.
White 96 well pcr plate
Manufactured by Bio-Rad
Sourced in United States
White 96-well PCR plates are a type of laboratory equipment used for performing polymerase chain reaction (PCR) experiments. These plates have 96 individual wells, each designed to hold a specific volume of sample and reagents for PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Cfx manager software, by Bio-Rad (2 mentions)
Optical quality sealing tape, by Bio-Rad (1 mentions)
Icycler iq5 multicolor real time pcr detection system, by Bio-Rad (1 mentions)
Cfx96 instrument, by Bio-Rad (1 mentions)
Mixmate, by Eppendorf (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Sypro orange, by Merck Group (1 mentions)
Cfx96 real time pcr cycler, by Bio-Rad (1 mentions)
6 protocols using white 96 well pcr plate
1
Thermal Shift Assay for MhGgH Melting
2
Thermal Shift Assay for UCHL1
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Reactions were performed in white 96-well PCR plates (Bio-Rad). UCHL1 was diluted in thermal shift buffer (1x PBS, 5 mM DTT) to a 4x concentration of 4 µM. The protein was then mixed with 4x compound dissolved in thermal shift buffer in a 1:1 ratio. To each well was added 20 µL of DUB-Inhibitor solution, followed by 1 h incubation time. To each well was then added 20 µL 4x SYPRO Orange in thermal shift buffer. After sealing the plates with a transparent film, thermal denaturation (gradient: 20–90 °C; Increment: 0.3 °C, hold for 5 s before read) was performed and monitored by a Bio-Rad Connect cycler.
3
Real-time RT-PCR Detection of FCoV RNA
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RNA was analyzed for presence of FCoV RNA by a real-time RT-PCR (RT-PCR) amplification of a well-conserved region of the membrane and nucleocapsid genes using primers (400 nM each) and probe sequences (200 nM, 5′ 6-FAM/ZEN/3′IB®FQ-labeled; Integrated DNA Technologies, Coralville, IA, USA), as previously described in [14 (link)] with a Luna Universal Probe One-Step RT-PCR reagent (New England Biolabs). Samples (5 µL or 0.5 µL of fecal RNA, or 100 ng of colon or PBMC RNA) were run in triplicate 20 µL reactions in white 96-well PCR plates (Bio-Rad, Hercules, CA, USA) using a Bio-Rad CFX96 instrument and the following cycling conditions: Reverse transcription (55 °C, 10 min), initial denaturation (95 °C, 1 min), 45 amplification cycles (95 °C 10 s, 60 °C 30 s), and final extension (72 °C, 10 min). Serial dilutions of FCoV-positive RNA were included on each plate for quality control. No FCoV RNA was detected in the buffer-only extraction controls or in the no-template controls included on each plate.
4
Differential Scanning Fluorimetry of QscR
5
Quantifying Bacterial Stress Tolerance
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Mutant cultures in stationary phase (20 μl) were put in a white 96-well PCR plate (Biorad), centrifuged, pellets were suspended in 100 μl of stress buffer. Phosphate buffer was supplemented with 170 g.L−1 ethanol for ethanol stress. Exposure to heat stress (52°C) was performed in prewarmed phosphate buffer and in a C1000™ Thermal Cycler (Biorad). After a 30-min incubation, bacterial pellets were suspended in 100 μl of 20 μM propidium iodide (PI, Sigma-Aldrich). All centrifugations were done at 4,000 g for 10 min and all suspensions were performed by vortexing (1 min at 1,550 rpm in a Mix mate, Eppendorf). Fluorescence (λem/λexc: 490/635 nm) was measured in a plate reader (Beckman Coulter) to calculate the viability percent with respect to a calibration range of known proportions of “dead” cells of L. paracasei WT (heat inactivated, 30 min at 80°C). Two biological replicates were made. When necessary, a third biological replicate was made to confirm the phenotype. Mutants were qualified as sensitive if their viability was less or equal to the WT viability minus twice the standard deviation for the two biological replicates. Corresponding genes were aligned using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) against all bacteria to determine their specificity.
6
Thermal-Shift Assay for NADP+ Binding
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Thermal-shift assays were performed on a CFX96 Real-Time PCR Cycler (Bio-Rad Laboratories). In these experiments, 1 μl of SYPRO Orange (Sigma-Aldrich, diluted from 5,000 × stock into Milli-Q), 8 μl protein (1.0 mg ml−1), and 1 μl buffer (1 M citrate buffer (pH 5.75)) were mixed on ice in a white 96-well PCR plate (Bio-Rad Laboratories). To evaluate the effect of NADP+ binding, 1 μl of 5,000 × SYPRO Orange, 8 μl protein (1.0 mg ml−1) and 1 μl buffer (1 M citrate buffer (pH 5.8)) containing 10 mM NADP+ were mixed. Fluorescence was measured as temperature increased from 25 to 85 °C in 0.5 °C steps (excitation, 450–490 nm; detection, 560–580 nm). All measurements were performed three times. Data evaluation and melting-point determination were conducted using the Bio-Rad CFX Manager software.
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