TITLE: Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles
AUTHORS: Jacob Beal, Traci Haddock-Angelli, Markus Gershater, Vishal Sanchania, Russell Buckley-Taylor, Geoff Baldwin, Natalie Farny, Richard Tennant, Paul Rutten

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>You will prepare a dilution series of ​monodisperse silica microspheres and measure the ​Abs​</span><span style = "vertical-align:sub;">600</span><span> in your plate reader. </span></div><div class = "text-block">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 600 nm absorbance measurements into an estimated equivalent number of cells.</div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [Prepare the Microsphere Stock Solution]
Obtain the tube labeled “Silica Beads” from the Measurement Kit and vortex vigorously for 30 seconds.
Microspheres should NOT be stored at 0°C or below, as freezing affects the properties of the microspheres. If you believe your microspheres may have been frozen, please contact the iGEM Measurement Committee for a replacement (measurement@igem.org).
?. [Prepare the Microsphere Stock Solution]
Immediately pipet 100 μL microspheres into a 1.5 mL eppendorf tube
?. [Prepare the Microsphere Stock Solution]
Add 900 μL of ddH2O​ to the microspheres
?. [Prepare the Microsphere Stock Solution]
Vortex well. This is your Microsphere Stock Solution
?. [Prepare the serial dilution of microspheres]
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. Column 12 must contain ddH​2O only. Initially you will setup the plate with the microsphere stock solution in column 1 and an equal volume of 1x ddH2O​ in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.
?. [Prepare the serial dilution of microspheres]
Add 100 μl of ddH​2O​ ​ into wells A2, B2, C2, D2....A12, B12, C12, D12
?. [Prepare the serial dilution of microspheres]
Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
?. [Prepare the serial dilution of microspheres]
Immediately add 200 μl​ ​of microspheres stock​ ​solution into A1
?. [Prepare the serial dilution of microspheres]
Transfer 100 μl of microsphere stock solution from A1 into A2
?. [Prepare the serial dilution of microspheres]
Mix A2 by pipetting up and down 3x and transfer 100 μl into A3
?. [Prepare the serial dilution of microspheres]
Mix A3 by pipetting up and down 3x and transfer 100 μl into A4
?. [Prepare the serial dilution of microspheres]
Mix A4 by pipetting up and down 3x and transfer 100 μl into A5
?. [Prepare the serial dilution of microspheres]
Mix A5 by pipetting up and down 3x and transfer 100 μl into A6
?. [Prepare the serial dilution of microspheres]
Mix A6 by pipetting up and down 3x and transfer 100 μl into A7
?. [Prepare the serial dilution of microspheres]
Mix A7 by pipetting up and down 3x and transfer 100 μl into A8
?. [Prepare the serial dilution of microspheres]
Mix A8 by pipetting up and down 3x and transfer 100 μl into A9
?. [Prepare the serial dilution of microspheres]
Mix A9 by pipetting up and down 3x and transfer 100 μl into A10
?. [Prepare the serial dilution of microspheres]
Mix A10 by pipetting up and down 3x and transfer 100 μl into A11
?. [Prepare the serial dilution of microspheres]
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
?. [Prepare the serial dilution of microspheres]
Repeat dilution series for rows B, C, D
?. [Prepare the serial dilution of microspheres]
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 OD]
Measure OD600​ of all samples in instrument. Disable any path length correction setting on your instrument, if it has one.If you will be using your data in conjuction with measurements from the Fluorescence standard curve protocol, make sure you use the same instrument settings for both protocols.
?. [Measure OD]
Record the data in your notebook. Please note your standard curve should still work well even if a few of your measurements are saturing the instrument
?. [Measure OD]
Import data into this Excel sheet:
?. [Congratulations!]
You have now completed this calibration protocol