2018 InterLab: Form II

Form II: Calibration Measurements

Every team that participates in the 2018 iGEM InterLab study needs to fill in four forms. This is Form II.

This form is part of your InterLab submission, and will ask you questions about your plate reader instrument, as well as the protocols you used for your calibration measurements. Fill this in as best as you can.

This form follows the InterLab protocol, pages 1-8 from this URL:
http://2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf

The purpose of this form is to document your performance of the calibration measurements, and to note any deviations from the protocols you may have made. This form will not provide the detailed protocols, so you must refer to the link above in order to perform these experiments.

If you have any questions or problems filling in this form, please email us at measurement at igem dot org.

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* 1. Team Name

Please enter your team name below. Please make sure it's the same as your official team name, including any hyphens or other characters.

Instrument Information

The use of a plate reader is required. If you do not have access to a plate reader, consider collaborating with another team if possible.

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* 2. What instrument did you use during your measurements?

plate reader

flow cytometry

Other...

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* 3. Please provide the brand and model of your plate reader.
(e.g., Tecan Infinite 200)

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* 4. Can your plate reader measure both absorbance and fluorescence?

If not, please contact the measurement committee to discuss alternatives.

Yes, it reads both fluorescence and absorbance

No, it only reads absorbance

No, it only reads fluorescence

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* 5. Does your plate reader use top or bottom optics?

(i.e. does your plate reader read samples from the top of the plate or the bottom)

Top optic

Bottom optic

Other (please specify)

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* 6. If you are doing the flow cytometry optional measurement, please provide the equipment details (brand, model) below.

Calibration Protocols

CALIBRATION PROTOCOLS SHOULD BE COMPLETED BEFORE CELL MEASUREMENTS ARE TAKEN!

You will make three sets of unit calibration measurements: an OD₆₀₀ reference point, a particle standard curve, and a fluorescein standard curve. Before beginning these protocols, please ensure that you are familiar with the measurement modes and settings of your instrument. For all of these calibration measurements, you must use the same plate type and volumes that you will use in your cell-based assays. You must also use the same settings (e.g., filters or excitation and emission wavelengths) that you will use in your cell-based assays.

If you do not use the same plates, volumes, and settings, the calibration will not be valid.

Make sure to record all information about your instrument (checklist on page 1 of the protocol) as these will be required later when you document your experiment. If your instrument has variable temperature settings, the instrument temperature should be set to room temperature (approximately 20-25 C) for all measurements.

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* 7. What type of 96-well plates did you use for the calibration measurements?

black plates with transparent/clear bottom (preferred)

white plates with transparent/clear bottom

clear plates

Other (please specify)

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* 8. Did your plates have flat-bottomed or round-bottomed wells?

We strongly recommend teams to use flat-bottomed plates. This is of critical importance if you do a bottom reading with your plate reader.

Flat

Round

Other (please specify)

Calibration 1: Optical Density (OD₆₀₀) Reference Point - LUDOX

You will use LUDOX CL-X as a single point reference to obtain a ratiometric conversion factor to transform your absorbance data into a standard OD₆₀₀ measurement. YOU MUST THEREFORE TURN OFF PATHLENGTH CORRECTION. To measure your standard LUDOX Abs₆₀₀ you must use the same plates and volumes (use 100 uL for plate reader measurement) that you will use in your cell based assays.

Prepare a column of 4 wells with 100 uL 100% LUDOX and 4 wells containing 100 uL dH₂O, as outlined in the protocol document (Pages 2-4).

OD₆₀₀ Reference Point: Instrument Settings

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* 9. Did you use pathlength correction during measurement?

Yes

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* 10. What temperature setting did you use during the measurement?

OD₆₀₀ Reference Point:: Measurement

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* 11. Steps for LUDOX measurement in a 96-well plate

Please check off each step that you followed from the InterLab protocol. If you did anything differently or extra, please note that in the "Other" box.

The LUDOX tube can be found in the Measurement Kit.

Add 100 μl LUDOX 100 % into wells A1, B1, C1, D1

Add 100 μl of dH2O into A2, B2, C2, D2

Measure absorbance 600 nm of all samples in the measurement mode you plan to use for cell measurements.

Record the data in the table on the InterLab protocol (Page 3) or in your notebook.

Import data into the blue cells on the OD600 Reference Point tab in the InterLab Excel document

Other (please specify)

Calibration 2: Particle Standard Curve - Microsphere

You will prepare a dilution series of monodisperse silica microspheres and measure the Abs₆₀₀ in your 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 will allow you to construct a standard curve of particle concentration which can be used to convert Abs₆₀₀ measurements to an estimated number of cells.

Particle Standard Curve: Instrument Settings

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* 12. Did you use pathlength correction during measurement?

Yes

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* 13. What temperature setting did you use during the measurement?

Particle Standard Curve: Measurement

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* 14. Was your tube of Silica Beads (microspheres) frozen?

The microspheres should NOT be stored below 0˚C as the freezing affects properties of the microspheres. If you answered "yes," or believe your microspheres have been frozen, please contact the measurement committee for a replacement by emailing measurement AT igem DOT org.

Yes

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* 15. Part 1: Steps for preparing the microsphere stock solution

Please check off each step that you followed from the InterLab protocol. If you did anything differently or extra, please note that in the "Other" box.

Obtain the tube labeled “Silica Beads” from the Measurement 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.

Other (please specify)

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* 16. Part 2: Prepare the serial dilutions of Microspheres
Accurate pipetting is essential! Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

Please see the InterLab protocol, pages 5-6, for complete details.

Prepare serial dilution as instructed (see InterLab protocol pages 5-6)

IMPORTANT! Re-Mix (Pipette up and down) each row of your plate immediately before putting in the plate reader! (This is important because the beads begin to settle to the bottom of the wells within about 10 minutes, which will affect the measurements.)

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)

Other (please specify)

Calibration 3: Fluoresence standard curve - FIuorescein

In this protocol, you will prepare a dilution series of fluorescein in 4 replicates and measure the fluorescence in a 96 well plate in your plate reader or individually in cuvettes in a fluorimeter. This will allow your measurements to be converted from arbitrary fluorescence into units of "μM fluorescein / OD"

Before beginning this protocol ensure that you are familiar with the GFP settings and measurement modes of your instrument. The settings that you use should be exactly the same ones that you will use when measuring your cells (if you change them you will not be able to use this standard curve). If you aren't absolutely certain which you will use, it can be a good idea to repeat the measurement a number of times with different settings. You will then have a series of standard curves to choose from without needing to redo this protocol. Most important, it is necessary to use a number of settings that affect the sensitivity (principally gain and/or slit width). Be sure to also consider other options (orbital averaging, top/bottom optics). As before, TURN OFF path length correction if available.

Fluoresence Standard Curve: Instrument Settings

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* 17. Did you use pathlength correction during measurement?

Yes

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* 18. What gain setting did you use?

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* 19. Did you use a filter on your instrument?

Yes

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* 20. If you used a filter, what light wavelengths did it pass?

(e.g., 530 nm / 30 nm bandpass, 25-30nm width is recommended)

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* 21. Emission wavelength (520-530nm is recommended)

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* 22. Excitation wavelength (485nm is recommended)

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* 23. What temperature setting did you use during the measurement?

Fluoresence Standard Curve: Measurement

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* 24. Part 1: Prepare the Fluorescein stock solution

Note: it is important that the fluorescein is properly dissolved. To check dissolution after the incubation period, pipet the solution up and down and visually inspect the fluid in your pipette tip – if any particulates are visible in the pipette tip continue to incubate overnight. Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

Spin down fluorescein tube from the Measurement Kit to make sure pellet is at the bottom of tube.

Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1ml of 1xPBS

Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM. Add 100 μL of 10x fluorescein stock into 900 μL 1xPBS.

Other (please specify)

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* 25. Part 2: Prepare the serial dilutions of Fluorescein

Accurate pipetting is essential! Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

Please see the InterLab protocol, pages 7-8, for complete details.

Prepare serial dilution as instructed (see InterLab protocol)

Measure fluorescence of all samples in instrument

Record the data in your notebook

Import data into Excel sheet provided (fluorescein standard curve tab)

Other (please specify)

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* 1. Team NamePlease enter your team name below. Please make sure it's the same as your official team name, including any hyphens or other characters.

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* 2. What instrument did you use during your measurements?

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* 3. Please provide the brand and model of your plate reader.(e.g., Tecan Infinite 200)

Question Title

* 4. Can your plate reader measure both absorbance and fluorescence?If not, please contact the measurement committee to discuss alternatives.

Question Title

* 5. Does your plate reader use top or bottom optics?(i.e. does your plate reader read samples from the top of the plate or the bottom)

Question Title

* 6. If you are doing the flow cytometry optional measurement, please provide the equipment details (brand, model) below.

Question Title

* 7. What type of 96-well plates did you use for the calibration measurements?

Question Title

* 8. Did your plates have flat-bottomed or round-bottomed wells?We strongly recommend teams to use flat-bottomed plates. This is of critical importance if you do a bottom reading with your plate reader.

Question Title

* 9. Did you use pathlength correction during measurement?

Question Title

* 10. What temperature setting did you use during the measurement?

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* 11. Steps for LUDOX measurement in a 96-well platePlease check off each step that you followed from the InterLab protocol. If you did anything differently or extra, please note that in the "Other" box.The LUDOX tube can be found in the Measurement Kit.

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* 12. Did you use pathlength correction during measurement?

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* 13. What temperature setting did you use during the measurement?

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* 15. Part 1: Steps for preparing the microsphere stock solutionPlease check off each step that you followed from the InterLab protocol. If you did anything differently or extra, please note that in the "Other" box.

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* 16. Part 2: Prepare the serial dilutions of MicrospheresAccurate pipetting is essential! Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.Please see the InterLab protocol, pages 5-6, for complete details.

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* 17. Did you use pathlength correction during measurement?

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* 18. What gain setting did you use?

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* 19. Did you use a filter on your instrument?

Question Title

* 20. If you used a filter, what light wavelengths did it pass?(e.g., 530 nm / 30 nm bandpass, 25-30nm width is recommended)

Question Title

* 21. Emission wavelength (520-530nm is recommended)

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* 22. Excitation wavelength (485nm is recommended)

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* 23. What temperature setting did you use during the measurement?

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* 25. Part 2: Prepare the serial dilutions of FluoresceinAccurate pipetting is essential! Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.Please see the InterLab protocol, pages 7-8, for complete details.

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* 26. Additional informationIf you need more space to explain any adjustments to this protocol, please enter your comments in the box below.

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* 27. FeedbackPlease let us know any other thoughts or comments you have about the InterLab study experience.

* 1. Team Name

Please enter your team name below. Please make sure it's the same as your official team name, including any hyphens or other characters.

* 3. Please provide the brand and model of your plate reader.
(e.g., Tecan Infinite 200)

* 4. Can your plate reader measure both absorbance and fluorescence?

If not, please contact the measurement committee to discuss alternatives.

* 5. Does your plate reader use top or bottom optics?

(i.e. does your plate reader read samples from the top of the plate or the bottom)

* 8. Did your plates have flat-bottomed or round-bottomed wells?

We strongly recommend teams to use flat-bottomed plates. This is of critical importance if you do a bottom reading with your plate reader.

* 11. Steps for LUDOX measurement in a 96-well plate

Please check off each step that you followed from the InterLab protocol. If you did anything differently or extra, please note that in the "Other" box.

The LUDOX tube can be found in the Measurement Kit.

* 15. Part 1: Steps for preparing the microsphere stock solution

Please check off each step that you followed from the InterLab protocol. If you did anything differently or extra, please note that in the "Other" box.

* 16. Part 2: Prepare the serial dilutions of Microspheres
Accurate pipetting is essential! Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

Please see the InterLab protocol, pages 5-6, for complete details.

* 20. If you used a filter, what light wavelengths did it pass?

(e.g., 530 nm / 30 nm bandpass, 25-30nm width is recommended)

* 25. Part 2: Prepare the serial dilutions of Fluorescein

Accurate pipetting is essential! Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

Please see the InterLab protocol, pages 7-8, for complete details.

* 26. Additional information

If you need more space to explain any adjustments to this protocol, please enter your comments in the box below.

* 27. Feedback

Please let us know any other thoughts or comments you have about the InterLab study experience.