Cfx96 touch real time pcr machine

A single colony of each strain was cultured overnight in THYB at 37 °C, and resuspended in the same medium at an OD₆₀₀ of 0.01. Cells were harvested at mid-exponential (OD₆₀₀ = 0.4–0.6) and stationary (OD₆₀₀ = 1.2–1.4) phases and added to two volumes of RNAprotect (Qiagen 76506). Total RNA was then isolated using the RNeasy minikit (Qiagen 74104) and the samples were treated with Turbo DNase (Life Technologies AM1907) to remove any genomic DNA. Conversion of RNA to cDNA was performed using SuperScript VILO cDNA synthesis kit (Invitrogen 11754050). Reverse transcription-quantitative PCR (RT-qPCR) was performed using SYBR green master mix (Applied Biosystems 4309155) according to the manufacturer’s instructions and primers specific for thfT (thfT-qPCR-S1: 5’-GCC AGT GGG TCA GGT AAT TT-3’; thfT-qPCR-A1: 5’-GAC AGT GGT TTC CGG TAG AAG-3’) and gyrA (RTPCR-gyrA-fw: 5’-CGA CTT GTC TGA ACG CCA AA-3’; RTPCR-gyrA-rv: 5’- GTC AGC AAT CAA GGC CAA CA-3’)^{26 (link)}. qPCR was performed on a CFX96 Touch Real-Time PCR machine (BioRad) and analysed using CFX Manager software (BioRad). Relative gene expression was calculated using the threshold cycle (2^−ΔΔCt) method with gyrA as the reference housekeeping gene. All reactions were performed in triplicate from three independently isolated RNA samples for each strain.

Thermal denaturation assays of purified and reconstituted Cgr2 were prepared on ice in 0.2 mL skirted 96-well PCR plates (VWR, Radnor, PA) sealed with optical adhesive covers (Life Technologies, Woburn, MA). Each reaction contained 10 µg of purified or reconstituted Cgr2, Sypro Orange protein gel stain (Thermo Fisher Scientific, Waltham, MA) diluted 5000-fold, and buffer containing 100 mM buffering agent and 100 mM NaCl in a total volume of 30 µL. The following buffering agents were used: acetate/acetic acid for pH 4–6, HEPES for pH 7, Tris-HCl for pH 8–9, and glycine-NaOH for pH 10. For metal binding assays, metal salts (Sigma-Aldrich, St. Louis, MO) were dissolved in pH 8 buffer to generate 100 mM stock solutions and added to a final concentration of 48 µM (8 equivalents relative to Cgr2). Data was collected on a CFX96 Touch Real-Time PCR machine (Bio-Rad, Hercules, CA) using the ‘FRET’ filter setting with FAM excitation and HEX emission channels (485 nm and 556 nm respectively). The following temperature-scan protocol was used: 25°C for 30 s, then ramp from 25°C to 100°C at a rate of 0.1 °C/ s.

The PrimeScript^TM RT Reagent Kit (Vazyme, Nanjing, China) was used for RNA extraction from seed samples and cDNA synthesis via reverse transcription. The CFX96 Touch Real-Time PCR machine (Bio-Rad) was adopted for quantitative polymerase chain reaction (qPCR) procedure using the ChamQ SYBR qPCR Master Mix (Vazyme). Supplementary Table 2 presents all primers used in the present study; 18srRNA was used as the endogenous reference. The fold change (FC) in expression was calculated as follows: FC = EΔCt, where E indicates the mean gene amplification efficacy and ΔCt stands for the difference in mean Ct values (obtained from all duplicates). The values were presented in the manner of mean SD after normalization. The gene expression analysis was performed with four biological replicates, and each was made in three technical replicates.

After the treated CL4176 and CL802 worms were transferred from 16 to 25 °C for 36 h, the worms were collected in an M9 buffer for RNA extraction using an RNeasy mini kit (QIAGEN, Hilden, Germany) following the manufacturer’s protocol. The RNA levels were measured by nanodrop (NanoDrop™ 2000/2000c spectrophotometer, Thermo Scientific), and their purity was evaluated by UV absorbance (260/280 ratio). Then, cDNA was synthesized using iScript™ Reverse Transcription Supermix R (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR was performed in a 10 µL mixture solution of SsoFast™ EvaGreen^® Supermix with Low ROX (Bio-Rad, Hercules, CA, USA), nuclease-free water, specific primers, and the cDNA. The primer sequences used in this study are listed in Table 1. PCR reactions were run in a CFX96 Touch Real-time PCR machine (Bio-Rad, Hercules, CA, USA) under the following conditions: real-time PCR was started with 1 cycle of 95 °C for 30 s, followed by 44 cycles at 95 °C for 5 s, and 60 °C for 30 s, and then 75 °C for 30 s to stop the reaction. The expressing Cq values were calculated with equation 2^-ΔΔCq for evaluating the fold change in the expression of each gene. The expression levels of mRNA were normalized using the internal control actin-1 (act-1). Data were obtained from three independent experiments with approximately 1000 worms in each group (n: ~3000).