Team:BUCT-China/InterLab
Instrument
PerkinElmer EnSpite Multimode Plate Reader
Features: This instrument can measure both absorbance and fluorescence.
Calibration 1: OD600 Reference point
Materials:
a.1ml LUDOX CL-X (provided in kit)
b.ddH20 (provided by team)
c.96 well plate, black with clear flat bottom preferred (provided by team)
Method:
a. Add 100 μl LUDOX into wells A1, B1, C1, D1
b.Add 100 μl of dd H2O into wells A2, B2, C2, D2
c. Measure absorbance at 600 nm of all samples in the measurement mode our plan to use for cell measurements
d.Record the data in the table below or in our notebook
e.Import data into Excel sheet provided (OD600 reference point tab)
Results:
Calibration 2: Particle Standard Curve
Materials:
a.300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10^8 microspheres)
b. ddH20 (provided by team)
c.96 well plate, black with clear flat bottom preferred (provided by team)
Method:
Prepare the Microsphere Stock Solution:
a. Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex
vigorously for 30 seconds. NOTE: Microspheres should NOT be stored at 0°C or below, as freezing affects the properties of the microspheres. If you believe our microspheres may have been frozen, please contact the iGEM MeasurementCommittee for a replacement (measurement at igem dot org).
b.Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
c.Add 904 μL of ddH2O to the microspheres
d.Vortex well. This is your Microsphere Stock Solution.
Prepare the serial dilution of Microspheres:
a.Add 100 μl of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12
b.Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
c.Immediately add 200 μl of microspheres stock solution into A1
d.Transfer 100 μl of microsphere stock solution from A1 into A2.
e.Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
f.Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
g.Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
h.Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
i.Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
j.Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
k.Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
l.Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
m.Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
n.Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
o. Repeat dilution series for rows B, C, D
p.IMPORTANT! Re-Mix (Pipette up and down) each row of your plate immediately before putting in the plate reader!
q.Measure Abs600 of all samples in instrument
r. Record the data in our notebook
s. Import data into Excel sheet provided (particle standard curve tab)
Results:
Calibration 3: Fluorescence standard curve
Materials:
a.Fluorescein (provided in kit)
b.10ml 1xPBS pH 7.4
c.96 well plate, black with clear flat bottom preferred (provided by team)
Method:
Prepare the Microsphere Stock Solution:
a. Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
b. Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. [Note: it is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up and down and examine the solution in the pipette tip – if any particulates are visible in the pipette tip continue to mix the solution until they disappear.]
c.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
Prepare the serial dilution of Microspheres:
a.Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
b.Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
c. Transfer 100 μl of fluorescein stock solution from A1 into A2.
d.Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
e.Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
f.Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
......
g.Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
h. Repeat dilution series for rows B, C, D
p. Re-Mix (Pipette up and down) each row of your plate immediately before putting in the plate reader!
q. Measure fluorescence of all iamples in instrument
g. Record the data in our notebook
k. Import data into Excel sheet provided ((fluorescein standard curve tab)
Results:
Cell measurement protocol
Materials:
a.Competent cells (Escherichia coli strain DH5α)
b.LB (Luria Bertani) media
c.Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
d.50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light
e.Incubator at 37°C
f.1.5 ml eppendorf tubes for sample storage
g.Ice bucket with ice
h.Micropipettes and tips
i.96 well plate, black with clear flat bottom preferred (provided by team)
j.Devices (from Distribution Kit, all in pSB1C3 backbone)
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:
a. Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
b. Measure Abs600 of these 1:10 diluted cultures
c. Record the data in your notebook
d. Dilute the cultures further to a target Abs600 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).
e. Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 µL samples per time point, 32 samples total). Place the samples on ice.
f. Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
g. Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
h. At the end of sampling point you need to measure your samples (Abs600 and fluorescence measurement), see the below for details.
p. Record data in your notebook
q. Import data into Excel sheet provided (fluorescence measurement tab)
Measurement:
Samples should be laid out according to the plate diagram below. Pipette 100 µl of each sample into each well. From 500 µl samples in a 1.5 ml eppendorf tube, 4 replicate samples of colony #1 should be pipetted into wells in rows A, B, C and D. Replicate samples of colony #2 should be pipetted into wells in rows E, F, G and H. Be sure to include 8 control wells containing 100uL each of only LB+chloramphenicol on each plate in column 9, as shown in the diagram below. Set the instrument settings as those that gave the best results in your calibration curves (no measurements off scale). If necessary you can test more than one of the previously calibrated settings to get the best data (no measurements off scale). Instrument temperature should be set to room temperature (approximately 20-25 C) if your instrument has variable temperature settings.
Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures
This procedure can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly relatable to the cell concentration of the culture, i.e. viable cell counts per mL. This protocol assumes that 1 bacterial cell will give rise to 1 colony.
For the CFU protocol, you will need to count colonies for your two Positive Control (BBa_I20270) cultures and your two Negative Control (BBa_R0040) cultures.
Step 1: Starting Sample Preparation
Standard Tracks: This protocol will result in CFU/mL for 0.1 OD600. Your overnight cultures will have a much higher OD600 and so this section of the protocol, called “Starting Sample Preparation”, will give you the “Starting Sample” with a 0.1 OD600 measurement.
Step 2: Dilution Series Instructions
Standard Tracks:Do the following serial dilutions for your triplicate Starting Samples you prepared in Step 1. You should have 12 total Starting Samples - 6 for your Positive Controls and 6 for your Negative Controls
Step 3: CFU/mL/OD Calculation Instructions
Standard Tracks: Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated as "colonies x Final Dilution Factor = CFU/mL "
Citation
Most of the information on this page comes from the document :2018_InterLab_Plate_Reader_Protocol.