Team:Gifu/InterLab

• 
  Project
  • Abstract
  • Back ground
  • How to make sake
  • Catabolite repression
  • Gap-repair cloning
  • Terminatome
  • Experiments
  • Plasmid construction
  • Results
  • Conclusion
  • Discussion
  • Future work
  • Reference
• 
  Lab Note
  • Protocol
  • Calendar
  • InterLab
• 
  Parts
  • Basic Part
  • Composite Part
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• Human Practices
  • Human Practices
  • Integrated HP
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  About Us
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INTERLAB

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Introduction

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We joined InterLab Measurement Study in this year. Eight devices were transformed into DH5a E.coli cells. We measured fluorescence of transformed cells.

Materials and Methods

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Materials

・Safire ART-NR:F129013

It is a plate reader to measure absorbance and fluorescence of samples.

Methods

1. Calculate the OD₆₀₀ Reference point

Measure absorbance 600 nm of LUDOX-S40 and H₂O in a 96 well plate in our plate reader. It is to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD₆₀₀ measurement.

2. Generate the FITC fluorescence standard curve

Prepare a dilution series of FITC in 4 replicates and measure the fluorescence in a 96 well plate in our plate reader. By measuring these in all standard modes, we can generate a standard curve of fluorescence for FITC concentration.

3. Measure cell

Day1 : Transform Escherichia coli these plasmids.
Plasmids
・Positive control
・Negative control
・Device1 : J23101.BCD2.E0040.B0015
・Device2：J23106.BCD2.E0040.B0015
・Device3：J23117.BCD2.E0040.B0015
・Device4：J23101+I13504
・Device5：J23106+I13504
・Device6：J23117+I13504

Day2 : Pick 2 colonies from each of plate and glow the cells in 5 ml LB medium + Chloramphenicol overnight. (37^○C and 220ppm)

Day3 : Cell growth, sampling, and assay
Measure OD₆₀₀ of these cells cultures and dilute it to a target OD₆₀₀ of 0.02 in 12 mL LB medium + Chloramphenicol and incubate the cultures (37^○C and 220rpm).At 0,2,4 and 6 hours of incubation take 500 μL samples of the cultures. At the end of sampling point, measure Absorbance 600 nm and Fluorescence of these samples. (Use same instrument setting when measuring the FITC standard curve.)

Result

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1. Calculate the OD₆₀₀ Reference point

The Abs₆₀₀ data of LUDOX and H₂O is Table1. And correction factor to transform our absorbance data into a standard OD₆₀₀ measurement is calculated by the following formula.

Table 1. Absorbance 600 nm of LUDOX-S40 and H₂O

2. Generate the FITC fluorescence standard curve

The fluorescence data of FITC is table2. And FITC standard curve is figure1.

Table 2. fluorescence data of FITC in any dilution

Fig.1 fluorescence standard curve

3. Measure cell

Table 3. blank substraction and correction (bacteria concentration)

Fig.2 blank substraction and correction (bacteria concentration)

Table 4. blank substraction (fluorescence)

Fig.3 blank substraction (fluorescence)

Table 5. Value of uM Fluorescein / OD₆₀₀

Fig.4 Value of uM Fluorescein / OD₆₀₀