| Line 106: | Line 106: | ||
</a> | </a> | ||
</li> | </li> | ||
| − | <li class="col-sm-2 col-xs-12" style="padding-left: 30px;"> | + | <li class="col-md-15 col-sm-2 col-xs-12" style="padding-left: 30px;"> |
<a href="#theme"> | <a href="#theme"> | ||
<div class="list-item"> | <div class="list-item"> | ||
❏ Add 100μL LUDOX into wells A1, B1, C1, D1
❏ Add 100μL of dd H2O 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)
❏ Obtain the tube labeled ”Silica Beads” from the InterLab test kit and vortex vigorously for 30 seconds.
❏ Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
❏ Add 904μL of ddH2O to the microspheres
❏ Vortex well. This is your Microsphere Stock Solution.
❏ Repeat dilution series for rows B, C, D
❏ Re-Mix (Pipette up and down) each row of plate immediately before putting in the plate reader
❏ Measure Abs600 of all samples in instrument
❏ Record the data in your notebook
❏ Import data into Excel sheet provided (particle standard curve tab)
❏ 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
❏ 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
❏ Measure fluorescence of all samples in instrument
❏ Record the data in your notebook
❏ Import data into Excel sheet provided (fluorescein standard curve tab)
❏ Resuspend DNA in selected wells in the Distribution Kit with 10µL dH20.
❏ Thaw competent cells on ice
❏ Pipette 50µL of competent cells into 1.5mL tube
❏ Pipette 1µL of resuspended DNA into 1.5mL tube
❏ Pipette 1µL of control DNA into 2mL tube
❏ Close 1.5mL tubes, incubate on ice for 30min
❏ Heat shock tubes at 42°C for 45 sec
❏ Incubate on ice for 5min
❏ Pipette 950µL SOC media to each transformation
❏ Incubate at 37°C for 1 hours, shaking at 200-300rpm
❏ Pipette 100µL of each transformation onto petri plates
❏ Spin down cells at 6800g for 3mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto petri plates
❏ Incubate transformations overnight (14-18hr) at 37°C
❏ Pick single colonies for PCR
❏ Count colonies for control transformation
❏ 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
❏ Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
❏ Measure Abs600 of these 1:10 diluted cultures
❏ Record the data in your notebook
❏ 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).
❏ 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.
❏ Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
❏ Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 mL eppendorf tubes. Place samples on ice.
❏ At the end of sampling point you need to measure your samples (Abs600 and fluorescence measurement), see the below for details.
❏ Record data in your notebook
❏ Import data into Excel sheet provided (fluorescence measurement tab)
❏ culture colonies for two Positive Control (BBa_I20270) cultures and your two Negative Control (BBa_R0040) cultures for 16-18 hours
❏ Dilute the overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for each culture. Check the OD600 and make sure it is 0.1
❏ Aseptically spead plate 100 μL on LB + Cam plates for those Final Dilution Factor is 8 x 104 or 8 x 105 or 8 x 106
❏ Incubate at 37°C overnight and count colonies after 18-20 hours of growth
❏ Count the colonies on each plate with fewer than 300 colonies. Multiple the colony count by the Final Dilution Factor on each plate.
Competent cells (Escherichia coli strain DH5α)
1mL LUDOX CL-X (provided in kit)
300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 108 microspheres)
LB (Luria Bertani) media
Fluorescein (provided in kit)
10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
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)
Devices (from Distribution Kit, all in pSB1C3 backbone):
Negative control BBa_R0040
Positive control BBa_I20270
Test Device 1 BBa_J364000
Test Device 2 BBa_J364001
Test Device 3 BBa_J364002
Test Device 4 BBa_J364007
Test Device 5 BBa_J364008
Test Device 6 BBa_J364009
According to the Reference OD600, We calculate the final result:
OD600/Abs600=3.818
All cell density readings using this instrument with the same settings and volume can be converted to OD600, so that we can use this ratio to convert subsequent experimental data
We prepare a dilution series of monodisperse silica microspheres and measure the Abs600 in plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allows us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.
The final result:
mean of med-high levels=6.24E+08
We prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in plate reader. By measuring these we generate a standard curve of fluorescence for fluorescein concentration. We will be able to use this to convert our cell based readings to an equivalent fluorescein concentration.
The final result:
Mean uM fluorescein / a.u.=3.60E-05
MEFL / a.u.=2.17E+08
