TITLE: Standard iGEM Cell Measurement Protocol
AUTHORS: Traci Haddock-Angelli, Jacob Beal, Natalie Farny, Chris Workman, Geoff Baldwin, Russell Buckley-Taylor, Ari Dwijayanti, Kristin Ellis, Markus Gershater, Daisuke Kiga, John Marken, Vinoo Selvarajah, Marko Storch, Richard Tennant, Paul Rutten

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol from the 2018 iGEM InterLab study which tests various 'standard' parts from the Registry.</div></div>

[STEPS]
?. [Day 1]
Transform ​Escherichia coli​ DH5α with these following plasmids (all in pSB1C3)  ABCD1DevicePart NumberPlateLocation2Negative controlBBa_R0040Kit Plate 7Well 2D3Positive control BBa_I20270Kit Plate 7Well 2B4Test Device 1BBa_J364000Kit Plate 7Well 2F5Test Device 2BBa_J364001Kit Plate 7Well 2H6Test Device 3BBa_J364002Kit Plate 7Well 2J7Test Device 4BBa_J364007Kit Plate 7Well 2L8Test Device 5BBa_J364008Kit Plate 7Well 2N9Test Device 6BBa_J364009Kit Plate 7Well 2P
ABCD1DevicePart NumberPlateLocation2Negative controlBBa_R0040Kit Plate 7Well 2D3Positive control BBa_I20270Kit Plate 7Well 2B4Test Device 1BBa_J364000Kit Plate 7Well 2F5Test Device 2BBa_J364001Kit Plate 7Well 2H6Test Device 3BBa_J364002Kit Plate 7Well 2J7Test Device 4BBa_J364007Kit Plate 7Well 2L8Test Device 5BBa_J364008Kit Plate 7Well 2N9Test Device 6BBa_J364009Kit Plate 7Well 2P
Help Debugging Your Transformations:
?. [Day 2]
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.
?. [Day 3]
Make a 1:10 dilution of each overnight culture in LB with Chloramphenicol (0.5 mL of culture into 4.5 mL of LB with Chloramphenicol)
?. [Day 3]
Measure the Abs​600​ of these 1:10 diluted cultures
?. [Day 3]
Record the data in your notebook
?. [Day 3]
Dilute the cultures further to a target Abs6​00 of 0.02 in a final volume of ​12 mL LB medium with Chloramphenicol in a 50 mL falcon tube
Falcon tube should be amber, or covered with foil to block light
?. [Day 3]
Take 500 μL samples of the diluted cultures at 0 hours into 1.5 mL eppendorf tubes, prior to incubation. At each time point between 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.
?. [Day 3]
Incubate the remainder of the cultures at 37 °C and 220 rpm for 6 hours
?. [Day 3]
Take 500 μL samples of the cultures at 6 hours of incubation into 1.5 mL eppendorftubes. Place samples on ice.
?. [Abs600 and Fluorescence Measurement]
At the end of the experiment, you should have two plates to read. You will have one plate for each time point: 0 and 6 hours. On each plate you will read both fluorescence and absorbance.Samples should be laid out according to the plate diagram below.
?. [Abs600 and Fluorescence Measurement]
Pipette 100 μL of each sample into each well. From 500 μL samples in a 1.5 mL eppendorf tube:4 replicate samples of colony #1 should be pipetted into wells in rows A, B, C and DReplicate samples of colony #2 should be pipetted into wells in rows E, F, G and H
?. [Abs600 and Fluorescence Measurement]
Be sure to include 8 control wells containing 100 μL each of only LB with Chloramphenicol on each plate in column 9, as shown in the diagram.Set the instrument settings as those that gave the best results in your calibration curves (no measurements off scale). If necessary you can test more than one of the previously calibrated settings to get the best data (no measurements off scale).
Instrument temperature should be set to room temperature (approximately 20-25 C) if your instrument has variable temperature settings
?. [Congratulations!]
You have now completed this calibration protocol