Recommendations for initial OD, culture volume and sealing the plate
- Our standard starting OD595/600 is 0.0625 (as measured using a 10mm path length standard cuvette). Higher or lower inoculation ODs are acceptable, but the start OD is important for several downstream applications.
- Using a start OD of 0.0625, 5 generations of growth are reached at the set OD generation point. The OD generation point is set as the reader OD of a 2 OD culture (measured in a 10 mm cuvette) and is determined from OD calibration curves (see the section OD calibration).
- The calculated growth metrics in the ACCESS software are computed to 5 generations using the OD generation point.
- For saving samples (see section well to well inoculation) experiments starting at OD595=0.0625 in 700 µl of media will allow 5 generations of growth at the time of save. For most growth conditions cells will be in the exponential growth regime at this point.
96 well: 100 µl (maximum volume assayes without well cross contamination is 120 µl)
48 well: 700 µl
384 well: 30-40µl
Note: 384 well plates need to be centrifuges (5 min at 2000 rpm) to ensure media is not wicked up the side of the well and to remove any air bubbles prior to initiating the experiment.
- After filling the wells, seal the plate with a film (Cat No AB-0580, ABGene). Make sure the film covers all the wells to avoid evaporation.
- Make sure there are no creases in the film, this will distort the measurements.
- Discard the plate lid.
- Do not damage or touch the bottom of plate or the film because the plate reader measures through bottom of plate
- Take care not to splash media on the adhesive seal, if splashing does occur replace the seal.
Pause point: the plate can be prepared and stored overnight at 4°C, if the strains are not temperature sensitive and the drug is stable in the medium.