Difference between revisions of "Team:NTHU Taiwan/InterLab"
Markswayne (Talk | contribs) |
|||
| Line 2: | Line 2: | ||
<html> | <html> | ||
| − | < | + | <p> |
| − | + | InterLab Study | |
| − | + | </p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| + | <p> | ||
| + | In this year’s InterLab study, our team has followed the InterLab_2017_Plate_Reader_Protocol to conduct the experiment. The work can be separated into two parts:(1) Calibration (2) Cell Measurement. | ||
</p> | </p> | ||
| − | </ | + | |
| + | <p> | ||
| + | (1) Calibration | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | a. OD 600 Reference point | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Materials: | ||
| + | -1ml LUDOX | ||
| + | -dd water | ||
| + | -96 well plates, clear with a flat bottom | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Use LUDOX-S40 as a single point reference to obtain a ratiometric conversion factor to transform the absorbance data into a standard OD 600 measurement. [*The workflow and protocol detail are presented in the below pdf.] | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | We will get the data for OD 600 of the H 2 O and LUDOX. The corrected Abs 600 is calculated by subtracting the H 2 O reading. To convert measured Abs 600 to OD 600 is to let Reference OD 600 divided by Abs 600. | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | b. Protocol fluorescein fluorescence standard curve | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Materials: | ||
| + | -Fluorescein | ||
| + | -10ml 1xPBS | ||
| + | -96 well plates, clear with a flat bottom | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Prepare a dilution series of fluorescein in 4 replicates and measure the fluorescence in a 96 well plate in the plate reader. Generate a standard curve of fluorescence for fluorescein concentration and use this to correct the cell-based readings to an equivalent fluorescein concentration. We must (1) Prepare the fluorescein stock solution (2) Prepare the serial dilutions of fluorescein. [*See pdf. for detail] | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Get “OD 600 reference point” & “Fluorescein standard curve” sheet. [*See the Excel Sheet 1&2 presented below] | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | (2) Cell Measurement | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Materials: | ||
| + | -Competent cells (Escherichia coli strain DH5α) | ||
| + | -LB (Luria Bertani) media | ||
| + | -Chloramphenicol (working stock 25 ug/mL) | ||
| + | -50 ml Falcon tube | ||
| + | -Incubator at 37°C | ||
| + | -1.5 ml Eppendorf tubes | ||
| + | -Ice bucket with ice | ||
| + | -Pipettes | ||
| + | -Devices (from InterLab Measurement Kit): | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Positive control | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Negative control | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Test Device 1: J23101+I13504 | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Test Device 2: J23106+I13504 | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Test Device 3: J23117+I13504 | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Test Device 4: J23101.BCD2.E0040.B0015 | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Test Device 5: J23106.BCD2.E0040.B0015 | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Test Device 6: J23117.BCD2.E0040.B0015 | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Day 1: Transform Escherichia coli DH5α with the Devices | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Day 2: Pick 2 colonies from each of plate and grow the cells overnight | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Day 3: Cell growth, sampling, and assay [*See pdf. for detail] | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | Layout for Abs600 and Fluorescence measurement (one plate for each time point: 0, 2, 4, and 6 hours). Get “Raw Plate Reader Measurements” & “Fluorescence Measurement” sheet. [See the Excel Sheet 3&4 presented below] | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | The experiment went quite smoothly, however, some of the “Summary Statistics” in the Fluorescence Measurement sheet cannot be calculated due to the negative value we’ve got from blanks in Fluorescence – Background. We think that it might be the mistake (ex. wrong concentration, mistakes in the volume or different gain number…) when we’re measuring the reference point, causing the background being larger than the sample OD value. | ||
| + | </p> | ||
| + | |||
Revision as of 12:59, 28 October 2017
NTHU_Taiwan
InterLab Study
In this year’s InterLab study, our team has followed the InterLab_2017_Plate_Reader_Protocol to conduct the experiment. The work can be separated into two parts:(1) Calibration (2) Cell Measurement.
(1) Calibration
a. OD 600 Reference point
Materials: -1ml LUDOX -dd water -96 well plates, clear with a flat bottom
Use LUDOX-S40 as a single point reference to obtain a ratiometric conversion factor to transform the absorbance data into a standard OD 600 measurement. [*The workflow and protocol detail are presented in the below pdf.]
We will get the data for OD 600 of the H 2 O and LUDOX. The corrected Abs 600 is calculated by subtracting the H 2 O reading. To convert measured Abs 600 to OD 600 is to let Reference OD 600 divided by Abs 600.
b. Protocol fluorescein fluorescence standard curve
Materials: -Fluorescein -10ml 1xPBS -96 well plates, clear with a flat bottom
Prepare a dilution series of fluorescein in 4 replicates and measure the fluorescence in a 96 well plate in the plate reader. Generate a standard curve of fluorescence for fluorescein concentration and use this to correct the cell-based readings to an equivalent fluorescein concentration. We must (1) Prepare the fluorescein stock solution (2) Prepare the serial dilutions of fluorescein. [*See pdf. for detail]
Get “OD 600 reference point” & “Fluorescein standard curve” sheet. [*See the Excel Sheet 1&2 presented below]
(2) Cell Measurement
Materials: -Competent cells (Escherichia coli strain DH5α) -LB (Luria Bertani) media -Chloramphenicol (working stock 25 ug/mL) -50 ml Falcon tube -Incubator at 37°C -1.5 ml Eppendorf tubes -Ice bucket with ice -Pipettes -Devices (from InterLab Measurement Kit):
Positive control
Negative control
Test Device 1: J23101+I13504
Test Device 2: J23106+I13504
Test Device 3: J23117+I13504
Test Device 4: J23101.BCD2.E0040.B0015
Test Device 5: J23106.BCD2.E0040.B0015
Test Device 6: J23117.BCD2.E0040.B0015
Day 1: Transform Escherichia coli DH5α with the Devices
Day 2: Pick 2 colonies from each of plate and grow the cells overnight
Day 3: Cell growth, sampling, and assay [*See pdf. for detail]
Layout for Abs600 and Fluorescence measurement (one plate for each time point: 0, 2, 4, and 6 hours). Get “Raw Plate Reader Measurements” & “Fluorescence Measurement” sheet. [See the Excel Sheet 3&4 presented below]
The experiment went quite smoothly, however, some of the “Summary Statistics” in the Fluorescence Measurement sheet cannot be calculated due to the negative value we’ve got from blanks in Fluorescence – Background. We think that it might be the mistake (ex. wrong concentration, mistakes in the volume or different gain number…) when we’re measuring the reference point, causing the background being larger than the sample OD value.
