Oct 11, 2020

Public workspaceCharacterization V.1

This protocol is a draft, published without a DOI.
  • 1Chung Shan Medical University
Cheng-Ruei Yang
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Protocol Citation: Cheng-Ruei Yang, Huan Jui Chang, iGEM-CSMU 2020. Characterization. protocols.io https://protocols.io/view/characterization-binjkdcn
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: July 16, 2020
Last Modified: October 11, 2020
Protocol Integer ID: 39339
Before start
pH buffer preparing(in 15 ml centrifuge tubes):
pH2: 5 ml ddH2O, 8.8 ul 35% HCl, 37.5 mg KCl
pH5: 5 ml ddH2O, 20.8 ul 99.7% CH3COOH, 162 ul 3M CH3COOK, 31 ul 10N NaOH
pH7: 5 ml RIPA buffer, 1.5 ul 35%HCl
pH8: 5 ml RIPA buffer, 1 ul 10N NaOH
pH10: 5 ml ddH2O, 0.0147 g NaHCO3, 8.8 ul NaOH
pH12: 5 ml ddH2O, 32.4 ul 10N NaOH, 0.06 g KCl

In vitro protein synthesis
In vitro protein synthesis
Add the reagents(on ice) for synthesis of mRFP into one well of the 384-well plate. Add the reagents from top to down.
OrderLocationReagentAmount
1Upper-leftDNase/RNase free waterTill 30 μL
2Upper-leftSolution A12μL
3Lower-rightSolution B9μL
4Lower-rightRNase inhibitor1.2μL
5Lower-leftRFP+terminator300ng
total30μL
Do triple repeat
Note
Pipetting Solution A, Solution B, RNase inhibitor before adding.
Pipetting the eppendorf contain RFP+terminator template to prevent precipitation.

Seal the plate with microseal (on ice).
Centrifuge Centrifigation4000 rpm, 4°C, 00:01:00

Setup plate reader 1
Setup plate reader 1
Plate type: 384 well plate
Select wells: At run time
Description: 1.TemperatureTemperature37 °C
Incubation
Incubation
Put the plate into plate reader.
Incubate the mixture and measure the fluorescence in plate reader follow Setup 1 for Duration02:00:00

Addition of Buffer
Addition of Buffer
Take the 384 well plate out of the plate reader.
Centrifuge Centrifigation4000 rpm, 4°C, 00:01:00
Remove its seal.
Apportion 5 μL of mRFP reagent into 18 well.
Note
Pipetting before apportion.
We actually add 4 μL into every well since we think it may evaporate.

Add pH2,5,7,8,10,12 Buffer solution respectively into different wells of mRFP till total volume of 100 μL.
Do triple repeat.
Note
Pipetting after adding buffer solution

Seal the well.
Centrifuge Centrifigation4000 rpm, 4°C, 00:01:00
Setup Plate Reader 2
Setup Plate Reader 2
Plate type: 384 well plate
Temperature: Temperature37 °C

Excitation start: 550nm, Stop: 620 nm
Fixed emission: 650 nm
Step: by 1 nm
Gain: Auto Gain (measured with selected wells)

Emission start: 550nm, Stop: 700 nm
Fixed excitation: 540nm
Step: by 1 nm
Gain: Auto Gain (measured with selected wells)
Measuring
Measuring
Measure the fluorescence excitation and emission intensity of mRFP