Difference between revisions of "Team:Purdue/Protocols"
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1. OD600 Reference point | 1. OD600 Reference point | ||
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You will use LUDOX-S30 as a single point reference to obtain a ratiometric conversion factor to | You will use LUDOX-S30 as a single point reference to obtain a ratiometric conversion factor to | ||
transform your absorbance data into a standard OD600 measurement. This has two key objectives.With standard 1 cm pathlength spectrophotometers, the reading is still instrument dependent (see above). With plate readers the path length is less than 1 cm and is volume dependent. In this instance the ratiometric conversion can both transform Abs600 measurements (i.e. the basic output of the instrument and not standardised optical density with 1 cm pathlength) into OD600 measurements, whilst simultaneously accounting for instrument differences. | transform your absorbance data into a standard OD600 measurement. This has two key objectives.With standard 1 cm pathlength spectrophotometers, the reading is still instrument dependent (see above). With plate readers the path length is less than 1 cm and is volume dependent. In this instance the ratiometric conversion can both transform Abs600 measurements (i.e. the basic output of the instrument and not standardised optical density with 1 cm pathlength) into OD600 measurements, whilst simultaneously accounting for instrument differences. | ||
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[IMPORTANT NOTE: many plate readers have an automatic path length correction, this is based on volume adjustment using a ratio of absorbance measurements at 900 and 950 nm. Because scattering increases with longer wavelengths, this adjustment is confounded by scattering solutions, such as dense cells. YOU MUST THEREFORE TURN OFF PATHLENGTH CORRECTION.] | [IMPORTANT NOTE: many plate readers have an automatic path length correction, this is based on volume adjustment using a ratio of absorbance measurements at 900 and 950 nm. Because scattering increases with longer wavelengths, this adjustment is confounded by scattering solutions, such as dense cells. YOU MUST THEREFORE TURN OFF PATHLENGTH CORRECTION.] | ||
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To measure your standard LUDOX Abs600 you must use the same cuvettes, plates and volumes (suggestion: use 100 μl for plate reader measurement and 1 mL for spectrophotometer measurement) that you will use in your cell based assays. The LUDOX solution is only weakly scattering and so will give a low absorbance value. | To measure your standard LUDOX Abs600 you must use the same cuvettes, plates and volumes (suggestion: use 100 μl for plate reader measurement and 1 mL for spectrophotometer measurement) that you will use in your cell based assays. The LUDOX solution is only weakly scattering and so will give a low absorbance value. | ||
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If using plates prepare a column of 4 wells with 100 μl 100% LUDOX and 4 wells containing 100 μl H2O. Repeat the measurement in all relevant modes used in your experiments (e.g. settings for orbital averaging). | If using plates prepare a column of 4 wells with 100 μl 100% LUDOX and 4 wells containing 100 μl H2O. Repeat the measurement in all relevant modes used in your experiments (e.g. settings for orbital averaging). | ||
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If using a cuvette, you will only have enough material for a single measurement, but repeat the reading multiple times. Use the same cuvette to measure the reference with H2O (this value will be subtracted by the instrument to give the OD600 reading). | If using a cuvette, you will only have enough material for a single measurement, but repeat the reading multiple times. Use the same cuvette to measure the reference with H2O (this value will be subtracted by the instrument to give the OD600 reading). | ||
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Materials: | Materials: | ||
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| − | 1ml LUDOX (provided in kit) | + | 1ml LUDOX (provided in kit)<br> |
| − | H20 (provided by team) | + | H20 (provided by team)<br> |
96 well plate or cuvettes (provided by team) | 96 well plate or cuvettes (provided by team) | ||
| − | + | <br> | |
Method | Method | ||
| − | + | <br> | |
Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette) | Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette) | ||
| − | Add 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette) | + | <br>Add 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette) |
| − | Measure absorbance 600 nm of all samples in all standard measurement modes in instrument | + | <br>Measure absorbance 600 nm of all samples in all standard measurement modes in instrument |
| − | Record the data in the table below or in your notebook | + | <br>Record the data in the table below or in your notebook |
| − | Import data into Excel (OD600 reference point tab) Sheet_1 provided | + | <br>Import data into Excel (OD600 reference point tab) Sheet_1 provided |
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2. Protocol FITC fluorescence standard curve | 2. Protocol FITC fluorescence standard curve | ||
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You will prepare a dilution series of FITC in 4 replicates and measure the fluorescence in a 96 well | You will prepare a dilution series of FITC in 4 replicates and measure the fluorescence in a 96 well | ||
plate in your plate reader or individually in cuvettes in a fluorimeter. By measuring these in all standard modes in your plate reader or fluorimeter, you will generate a standard curve of fluorescence for FITC concentration. You will be able to use this to correct your cell based readings to an equivalent fluorescein concentration. You will then be able to convert this into a concentration of GFP. | plate in your plate reader or individually in cuvettes in a fluorimeter. By measuring these in all standard modes in your plate reader or fluorimeter, you will generate a standard curve of fluorescence for FITC concentration. You will be able to use this to correct your cell based readings to an equivalent fluorescein concentration. You will then be able to convert this into a concentration of GFP. | ||
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Before beginning this protocol ensure that you are familiar with the GFP settings and measurement modes of your instrument. | Before beginning this protocol ensure that you are familiar with the GFP settings and measurement modes of your instrument. | ||
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| + | <br> | ||
Materials: | Materials: | ||
| − | + | <br> | |
187 μg FITC (provided in kit) | 187 μg FITC (provided in kit) | ||
| − | 10ml 1xPBS (phosphate buffered saline; provided by team) | + | <br>10ml 1xPBS (phosphate buffered saline; provided by team) |
| − | 96 well plate or cuvettes (provided by team) | + | <br>96 well plate or cuvettes (provided by team) |
| − | + | <br> | |
| + | <br> | ||
Method | Method | ||
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Prepare the FITC stock solution: | Prepare the FITC stock solution: | ||
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Spin down FITC stock tube to make sure pellet is at the bottom of tube. | Spin down FITC stock tube to make sure pellet is at the bottom of tube. | ||
| − | Prepare 10x FITC stock solution by resuspending FITC in 1 mL of 1xPBS | + | <br>Prepare 10x FITC stock solution by resuspending FITC in 1 mL of 1xPBS |
| − | Incubate the solution at 42°C for 4 hours | + | <br>Incubate the solution at 42°C for 4 hours |
| − | Dilute the 10x FITC stock solution in half with 1xPBS to make a 5x FITC solution and resulting concentration of FITC stock solution 2.5 μM. | + | <br>Dilute the 10x FITC stock solution in half with 1xPBS to make a 5x FITC solution and resulting concentration of FITC stock solution 2.5 μM. |
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[Note: it is important that the FITC is properly dissolved. To check this after the incubation period pipetted up and down – if any particulates are visible in the pipette tip continue to incubate overnight.] | [Note: it is important that the FITC is properly dissolved. To check this after the incubation period pipetted up and down – if any particulates are visible in the pipette tip continue to incubate overnight.] | ||
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Prepare the serial dilutions of FITC: | Prepare the serial dilutions of FITC: | ||
| − | + | <br> | |
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN PBS BUFFER ONLY. Initially you will setup the plate with the FITC stock in column 1 and an equal volume of 1xPBS in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing. | Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN PBS BUFFER ONLY. Initially you will setup the plate with the FITC stock in column 1 and an equal volume of 1xPBS in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing. | ||
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Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12 | Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12 | ||
| − | Add 200 μl of FITC 5x stock solution into A1, B1, C1, D1 | + | <br>Add 200 μl of FITC 5x stock solution into A1, B1, C1, D1 |
| − | Transfer 100 μl of FITC stock solution from A1 into A2. | + | <br>Transfer 100 μl of FITC stock solution from A1 into A2. |
| − | Mix A2 by pipetting up and down 3x and transfer 100 μl into A3... | + | <br>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... | + | <br>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... | + | <br>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... | + | <br>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... | + | <br>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... | + | <br>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... | + | <br>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... | + | <br>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... | + | <br>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 | + | <br>Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste |
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TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12. | TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12. | ||
| − | + | <br> | |
Repeat dilution series for rows B, C, D | Repeat dilution series for rows B, C, D | ||
| − | Measure fluorescence of all samples in all standard measurement modes in instrument | + | <br>Measure fluorescence of all samples in all standard measurement modes in instrument |
| − | Record the data in your notebook | + | <br>Record the data in your notebook |
| − | Import data into Excel (FITC standard curve tab) Sheet_1 provide | + | <br>Import data into Excel (FITC standard curve tab) Sheet_1 provide |
</p> | </p> | ||
Revision as of 00:45, 14 October 2016
Protocols
Plate Reader Calibration Protocols
1. OD600 Reference point
You will use LUDOX-S30 as a single point reference to obtain a ratiometric conversion factor to
transform your absorbance data into a standard OD600 measurement. This has two key objectives.With standard 1 cm pathlength spectrophotometers, the reading is still instrument dependent (see above). With plate readers the path length is less than 1 cm and is volume dependent. In this instance the ratiometric conversion can both transform Abs600 measurements (i.e. the basic output of the instrument and not standardised optical density with 1 cm pathlength) into OD600 measurements, whilst simultaneously accounting for instrument differences.
[IMPORTANT NOTE: many plate readers have an automatic path length correction, this is based on volume adjustment using a ratio of absorbance measurements at 900 and 950 nm. Because scattering increases with longer wavelengths, this adjustment is confounded by scattering solutions, such as dense cells. YOU MUST THEREFORE TURN OFF PATHLENGTH CORRECTION.]
To measure your standard LUDOX Abs600 you must use the same cuvettes, plates and volumes (suggestion: use 100 μl for plate reader measurement and 1 mL for spectrophotometer measurement) that you will use in your cell based assays. The LUDOX solution is only weakly scattering and so will give a low absorbance value.
If using plates prepare a column of 4 wells with 100 μl 100% LUDOX and 4 wells containing 100 μl H2O. Repeat the measurement in all relevant modes used in your experiments (e.g. settings for orbital averaging).
If using a cuvette, you will only have enough material for a single measurement, but repeat the reading multiple times. Use the same cuvette to measure the reference with H2O (this value will be subtracted by the instrument to give the OD600 reading).
Materials:
1ml LUDOX (provided in kit)
H20 (provided by team)
96 well plate or cuvettes (provided by team)
Method
Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
Add 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
Record the data in the table below or in your notebook
Import data into Excel (OD600 reference point tab) Sheet_1 provided
2. Protocol FITC fluorescence standard curve
You will prepare a dilution series of FITC in 4 replicates and measure the fluorescence in a 96 well
plate in your plate reader or individually in cuvettes in a fluorimeter. By measuring these in all standard modes in your plate reader or fluorimeter, you will generate a standard curve of fluorescence for FITC concentration. You will be able to use this to correct your cell based readings to an equivalent fluorescein concentration. You will then be able to convert this into a concentration of GFP.
Before beginning this protocol ensure that you are familiar with the GFP settings and measurement modes of your instrument.
Materials:
187 μg FITC (provided in kit)
10ml 1xPBS (phosphate buffered saline; provided by team)
96 well plate or cuvettes (provided by team)
Method
Prepare the FITC stock solution:
Spin down FITC stock tube to make sure pellet is at the bottom of tube.
Prepare 10x FITC stock solution by resuspending FITC in 1 mL of 1xPBS
Incubate the solution at 42°C for 4 hours
Dilute the 10x FITC stock solution in half with 1xPBS to make a 5x FITC solution and resulting concentration of FITC stock solution 2.5 μM.
[Note: it is important that the FITC is properly dissolved. To check this after the incubation period pipetted up and down – if any particulates are visible in the pipette tip continue to incubate overnight.]
Prepare the serial dilutions of FITC:
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN PBS BUFFER ONLY. Initially you will setup the plate with the FITC stock in column 1 and an equal volume of 1xPBS in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.
Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
Add 200 μl of FITC 5x stock solution into A1, B1, C1, D1
Transfer 100 μl of FITC 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
TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.
Repeat dilution series for rows B, C, D
Measure fluorescence of all samples in all standard measurement modes in instrument
Record the data in your notebook
Import data into Excel (FITC standard curve tab) Sheet_1 provide
