Difference between revisions of "Team:HUST-China/InterLab"

a.Protocol

Use pathlength correction
Number of flashes per well：6
Orbital averaging (nm)：600
Temperature setting：22℃
Type of 96-well plate：Black plate (preferred) Our plates have flat-bottomed wells.

b.Measurement Steps

Add 100ul LUDOX into wells A1, B1, C1,D1
Add 100ul ddH2O 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
data into Excel sheet provided (OD600 reference point tab)

a.Protocol

Use pathlength correction
Number of flashes per well：6
Orbital averaging (nm)：600
Temperature setting：22℃
Type of 96-well plate：Black plate (preferred)
Our plates have flat-bottomed wells.

b.Measurement Steps

Prepare the Microsphere Stock Solution:
❏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 904 μL of ddH2O to the microspheres
❏Vortex well. This is your Microsphere Stock Solution.

❏Add 100 μl of ddH2O 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 μl of 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
❏ Repeat dilution series for rows B, C, D
❏Re-Mix each row of your plate immediately before putting in the plate reader! Take care to mix gently and avoid creating bubbles on the surface of the liquid.
❏ Measure Abs600 of all samples in instrument
❏ Record the data in your notebook
❏ Import data into Excel sheet provided (particle standard curve tab)

a.Protocol

Use pathlength correction
Number of flashes per well：  6
Gain setting:  Automatic
It passed 530nm when we used a filter.
Emission wavelength:   525nm
Excitation wavelength:   485nm
Fluorescence reading:   Bottom optic
Type of 96-well plate：   Black plate (preferred)
Our plates have flat-bottomed wells.
Temperature setting:   22℃

b.Measurement Steps

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

❏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)

Transformation:
Negative control BBa_R0040 Kit Plate 7 Well 2D
Positive control BBa_I20270 Kit Plate 7 Well 2B
Test Device 1 BBa_J364000 Kit Plate 7 Well 2F
Test Device 2 BBa_J364001 Kit Plate 7 Well 2H
Test Device 3 BBa_J364002 Kit Plate 7 Well 2J
Test Device 4 BBa_J364007 Kit Plate 7 Well 2L
Test Device 5 BBa_J364008 Kit Plate 7 Well 2N
Test Device 6 BBa_J364009 Kit Plate 7 Well 2P

Cell grow:
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.

Cell growth, sampling, and assay
 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
 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.
 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)
 Record data in notebook
 Import data into Excel sheet provided (fluorescence measurement tab)

 Type of media we used for this step: Luria Bertani
 Type of vessel or container we used to grow cells: 50 ml Falcon tube
 Temperature setting:22℃
 Type of 96-well plates: Black plates with transparent/clear bottom (preferred),flat
 Measurement: Measure OD and fluorescence of all samples
 Suggested Plate Layout for 96-well Plate