OD₆₀₀ Reference point

Materia
1ml LUDOX CL-X (provided in kit)
ddH​ 2​ 0 (provided by team)
96 well plate, black with clear flat bottom preferred (provided by team)


Method
Add 100 μl LUDOX into wells A1, B1, C1, D1
Add 100 μl of dd H​ 2​ O into wells A2, B2, C2, D2
Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements
Record the data in the table below or in your notebook
Import data into Excel sheet provided (​OD₆₀₀ reference point tab​​)

Results

Particle Standard Curve

Materia
300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10^8 microspheres)
ddH20 (provided by team) 96 well plate, black with clear flat bottom preferred (provided by team)

Method
Prepare:
Stock Solution
Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex
Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
vigorously for 30 seconds.
Add 904 μL of ddH2O to the microspheres
Vortex well.
serial dilution:
Add 100 μl of ddH20 into wells A2, B2, C2, D2....A12, B12, C12, D12
Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
Immediately add 200 μlof microspheres stock solution into A1
Transfer 100 μl of microsphere stock solution from A1 into A2.
Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
Mix A11 by pipetting up and down 3x and transfer 100 μl into ​liquid waste

Results

The standard curve of Fluorescein's fluorescence

Materia
Fluorescein (provided in kit)
10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
96 well plate, black with clear flat bottom (provided by team)

Method
Prepare the fluorescein stock solution:
Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS
Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein
Solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x PBS
serial dilutions:
Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
Transfer 100 μl of fluorescein stock solution from A1 into A2.
Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
Mix A11 by pipetting up and down 3x and transfer 100 μl into ​liquid waste
Repeat dilution series for rows B, C, D

Results

Cell measure

Materia
Competent cells (​Escherichia coli strain DH5α)
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
Incubator at 37°C
1.5 ml eppendorf tubes for sample storage
Ice bucket with ice
Micropipettes and tips
96 well plate, black with clear flat bottom preferred (provided by team)

Method
Day 1​​: transform ​Escherichia coli DH5α with these following plasmids (all in pSB1C3)
Day 2​​: Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
Day 3 and after​​: Cell growth, sampling, and assay
1、Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
2、Measure Abs​ 600​ of these 1:10 diluted cultures
3、Record the data in the notebook
4、 Dilute the cultures further to a target Abs​ 600 of 0.02 in a final volume of ​12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).
5、Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation.Place the samples on ice.
6、Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
7、 Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
8、At the end of sampling point you need to measure your samples (Abs​ 600 and fluorescence measurement), see the below for details.

Results