Ⅰ.Introduction

The goal of the interlab study is to explore a major question: How close can the numbers be when fluorescence is measured all around the world? So we measure GFP fluorescence in our lab with plate reader (Tecan Infinite M200pro). This year, three devices and one positive control and one negative control were provided by the registry. The results show increased fluorescence in the stronger promoter expected.

Digestion Verification

Ⅱ. Protocol

Cell measurement protocol

1. Transform five plasmids into DH5α competent cells, grown in incubator for 12 hrs at 37℃.
2. Pick 2 colonies from each of plate and inoculate it on 10mL LB medium+ Chloramphenicol. Grow the cells for 16hrs at 37°C and 220rpm.
3. Cell growth, sampling, and assay.
4. Obtain initial OD 600 measurement of overnight cultures. Then dilute each sample to an OD of 0.02.
5. Incubate the cultures at 37°C and 220rpm. Take 100µL (1% of total volume) samples of the cultures at 0, 1, 2, 3, 4, 5 and 6 hours of incubation.
6. At the end of sampling point measure these samples (OD and Fl measurement).
7. Measurements of absorbance and fluorescence:
(1) OD 600
Device: Plate Reader（Tecan Infinite M200pro） Wavelengths: 600 nm absorption.
(2) Fluorescence
Device: Plate Reader（Tecan Infinite M200pro）, 96-well plates. Wavelengths: 485 nm excitation, 520 nm emission.

Ⅲ. Measurements

1. OD600 Reference point

Table 1. Absorbance at 600nm for LUDOX and H2O

2. FITC standard curve

Table 2. Fluorescence for FITC at decreasing concentration

3. Normalisation

Table 3.  Absorbance at 600nm for five devices after cell growth for 16 hours and media+chl

4. Cell measurement

1. Abs600

Table 4.  Absorbance at 600nm for five devices after blank substraction and correction Click here to check raw data

Figure 4. The plot of Abs600 versus time

2. Fluorescence

Table 5.   Fluorescence for five devices after blank substraction Click here to check raw data

Figure 5.  The plot of FI versus time in microplate reader with 485nm excitation, 520nm emission

3. Fl/Abs600

Table 6.  The FI/Abs600 of five decives

Figure 6.  The chart of FI/Abs600 versus time for five devices

4. Average and SD

Table 7. The average and SD of fluorescence for five devices

Ⅳ. Provenance

Q: Who did the actual work to acquire these measurements?
A: Zeyue Gao.

Q: What other people should be credited for these measurements?
A: Kainan Chen.

Q: On what dates were the protocols run and the measurements taken?
A: Required devices were transformed on 14th August, 2016. All samples were measured on 15th August, 2016.