The pEVOL plasmid encoding tRNAProT and the sfGFP-mCherry reporter plasmid were transformed into E. coli MG1655 cells. Single colonies were used to inoculate LB medium containing Kan, Chl, and 0.2 mM IPTG to induce the expression of sfGFP-mCherry in a 96-well microplate. Cells were incubated for 24 h with constant shaking at 37 °C using a BioTek HTX plate reader. sfGFP (excitation 485 nm, emission 510 nm) and mCherry (excitation 587 nm, emission 610 nm) fluorescence and A600 were measured. For consistency, we chose the individual mid-exponential growth phase of E. coli for determining sfGFP-mCherry fluorescence and mistranslation frequency. This was accomplished by determining the time at which E. coli cells reached the mid-exponential growth phase using the preset function “Sigmoidal, 4P, X is concentration” in Prism 9 (GraphPad). The sfGFP and mCherry fluorescence proportion was calculated at the mid-exponential growth phase.
Htx plate reader
Manufactured by Agilent Technologies
Sourced in Germany
The HTX plate reader is a high-throughput microplate reader designed for a variety of assays. It can measure absorbance, fluorescence, and luminescence in microplates with up to 1536 wells.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using htx plate reader
1
Dual-reporter Assay for Protein Mistranslation
2
Analytical Methods for Metabolite Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Supernatants containing glucose and lactate were quantified using a Biosen C-line Analyzer (EKF Diagnostics, Barleben, Germany), according to the manufacturer’s protocol. Ammonia and glutamine titers were estimated with a K-GLNAM kit (Megazyme, Bray, Ireland), mostly according to manufacturer’s protocol, but extending the measuring time to 15 min for the second enzymatic reaction. The calibration ranges were set to 0.04–0.1 mg·mL−1 for ammonia and 0.1–0.7 mg·mL−1 for glutamine. The BSA concentration was determined using Roti-Nanoquant (Carl Roth, Karlsruhe, Germany) with a linear range of 2.5–100 µg·mL−1. For all kits, we used an HTX plate reader (BioTek, Friedrichshall, Germany) for absorption measurements. Dissolved oxygen levels were monitored with an OXY-4 mini meter (Presens, Regensburg, Germany) and OXY4 software (Version v2_11fb, 2011, Presens, Regensburg, Germany). For this purpose, we used an autoclavable flow-through cell setup (100 µL volume, Presens, Regensburg, Germany) equipped with an internal chemo-optical YAU-based sensor (Presens, Regensburg, Germany). The equipment was calibrated according to the manufacturer’s instructions at 25 °C.
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!