Oct 24, 2020
Improve
This protocol is a draft, published without a DOI.
- 1Chung Shan Medical University
- Chung Shan Medical University
Protocol Citation: Hung Yu Chen, Huan Jui Chang 2020. Improve . protocols.io https://protocols.io/view/improve-bnw2mfge
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 24, 2020
Last Modified: October 24, 2020
Protocol Integer ID: 43706
In vitro protIn vitro protein synthesis
In vitro protIn vitro protein synthesis
Add the reagents(on ice) for synthesis of amilCP into 15 wells of the 384-well plate. Pipette well before adding reagents into the wells.
banana amilCP
| Order | Location | Reagent | Amount | |
| 1 | Upper-left | DNase/ RNase free water | till 5 μL | |
| 2 | Upper-left | Solution A | 2 μL | |
| 3 | Lower-right | Solution B | 1.5 μL | |
| 4 | Lower-right | RNase inhibitor | 0.2 μL | |
| 5 | Lower-left | banana amilCP | 50 ng | |
| Total | 5 μL |
banana trigger + banana amilCP
| Order | Location | Reagent | Amount | |
| 1 | Upper-left | DNase/ RNase free water | till 5 μL | |
| 2 | Upper-left | Solution A | 2 μL | |
| 3 | Lower-right | Solution B | 1.5 μL | |
| 4 | Lower-right | RNase inhibitor | 0.2 μL | |
| 5 | Lower-left | banana amilCP | 50 ng | |
| 6 | Lower-left | banana trigger | 50 ng | |
| Total | 5 μL |
T7 Strong Promoter + amilCP
| Order | Location | Reagent | Amount | |
| 1 | Upper-left | DNase/ RNase free water | till 5μL | |
| 2 | Upper-left | Solution A | 2 μL | |
| 3 | Lower-right | Solution B | 1.5 μL | |
| 4 | Lower-right | RNase inhibitor | 0.2 μL | |
| 5 | Lower-left | T7 Strong Promoter amilCP | 50 ng | |
| Total | 10 μL |
Note
Do triple repeat
Seal the plate with microseal (on ice)
Centrifuge (4000rpm, 0.01 (1 min), 4°C)
Setup plate reader
Setup plate reader
Temperature: Setpoint 37°C
Shake: Orbital for 1:00 (1 min)
Start Kinetic [ Run 6:00:00 (6 hour), Internal 0:05:00 (5 min)]
Read: (A) 588 nm
End Kinetic
Incubation
Incubation
Put the plate into plate reader.
Incubate the mixture and measure the fluorescence in plate reader follow Setup 1 for 06:00:00
Measuring
Measuring
Measure the fluorescence excitation and emission intensity of the parts.