OKF6/Tert-1 cells were treated with either 1 μg/ml double-stranded RNA mimetic poly(I:C) (Invivogen, tlrl-picw) in cell culture media for up to six hours. RNA of cells was isolated using the RNEasy Plus Mini kit (Qiagen, 74136) and subsequently converted to cDNA via reverse transcription using the iScript Supermix (Bio-Rad, 1708840). qPCR was performed on the cDNA using SsoAdvanced SYBR Green (Bio-Rad, 172–5274) in the CFX96-C1000 Touch thermocycler (Bio-Rad). Relative expression of type I interferon or interferon-stimulated genes were measured using the 2−ΔΔCq calculations (Winer, Kwang, Jung, Shackel, & Williams, 1999 ) in the CFX Manager software (Bio-Rad) with ACTB serving as the reference gene. Primers sequences were generated using the PrimerQuest tool (IDT). The list of primers used in these experiments can be found in Table 1 .
Cfx96 c1000 touch thermocycler
Manufactured by Bio-Rad
The CFX96-C1000 Touch thermocycler is a real-time PCR instrument designed for versatile and efficient nucleic acid amplification and detection. It features a 96-well format and a touchscreen interface for user-friendly operation.
Automatically generated - may contain errors
Lab products found in correlation
Iscript supermix, by Bio-Rad (1 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Ssoadvanced sybr green, by Bio-Rad (1 mentions)
Sypro orange dye, by Thermo Fisher Scientific (1 mentions)
Cfx maestro software, by Bio-Rad (1 mentions)
Qscript xlt cdna supermix, by Quantabio (1 mentions)
Miscript sybr green pcr system, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr green one step kit, by Bio-Rad (1 mentions)
4 protocols using cfx96 c1000 touch thermocycler
1
Quantifying Type I Interferon Response
2
Thermal Shift Assay of Penicillin-Binding Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DSF thermal shift assay was conducted on a CFX96-C1000-Touch thermocycler (Bio-Rad) similarly to what we previously described (29 (link), 30 (link)). The reactions were carried out in the buffer containing 50 mM HEPES, pH 8.0, 300 mM NaCl, 10% glycerol at a sample volume of 30 μl, with 1.4 μM PBP2 either with or without WCK 5153 and zidebactam compounds dissolved in dimethyl sulfoxide (DMSO). Equivalent DMSO controls were also included, and reactions were run in duplicates. All the reaction mixtures included SYPRO orange dye (Fisher Scientific) at 10× concentration, and the thermal scan was conducted between 25 and 70°C with 0.2°C/min intervals.
For PBP3 (3 μM), the DSF experiments were conducted for all four DBO compounds (zidebactam, WCK 5153, WCK 4234, and avibactam) at the indicated concentrations. The first three DBOs were dissolved in DMSO, and DSF experiments were carried out in duplicate. Avibactam was dissolved in water, and DSF experiments with avibactam-containing mixtures were carried out in triplicate. Ceftazidime (at 200 μM) was used as a positive control similarly to what was previously described (29 (link), 30 (link)). The reaction buffer contained 14 mM Tris, pH 8.0, 280 mM NaCl, 7% glycerol, and 10× SYPRO orange.
For PBP3 (3 μM), the DSF experiments were conducted for all four DBO compounds (zidebactam, WCK 5153, WCK 4234, and avibactam) at the indicated concentrations. The first three DBOs were dissolved in DMSO, and DSF experiments were carried out in duplicate. Avibactam was dissolved in water, and DSF experiments with avibactam-containing mixtures were carried out in triplicate. Ceftazidime (at 200 μM) was used as a positive control similarly to what was previously described (29 (link), 30 (link)). The reaction buffer contained 14 mM Tris, pH 8.0, 280 mM NaCl, 7% glycerol, and 10× SYPRO orange.
3
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In total, 0.5–1 μg of total RNA extracted using the Qiagen Kit was reverse transcribed to generate cDNA using qScript XLT cDNA SuperMix (Quantabio). qPCR was performed on a CFX96 C1000 Touch Thermocycler, and the data were analyzed with CFX Maestro Software (Version 4.1.2433.1219, Bio-Rad). The thermocycling conditions are: (a) 95°C for 10 minutes, (b) 95°C for 15 seconds, (c) 60°C for 60 seconds, (d) plate read, (e) repeat steps b–d for 39 cycles, (f) 65°C for 30 seconds, and (g) hold at 4°C. 18s ribosomal RNA was used as the reference gene to normalize the data. Sequence-specific primers are listed in Supplemental Table 3 .
4
Quantifying miRNA Regulation in Cell Lines
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!