Griffitts:ß-Gal/GUS assay

Procedure

 * Grow cultures overnight to saturation
 * Sub-culture 1:10 into fresh media
 * Cells can be spun down and re-suspended in fresh media for increased experimental control or sub-cultured directly from saturated cultures
 * Sub-culturing can be done in triplicate
 * Let the sub-cultures grow for 3-4 hours
 * If you are planning on letting the sub-culture grow longer make the dilution greater
 * The cells need to be actively growing and have and OD600 of ~1.000 when assayed, so sub-culture accordingly
 * Take the culture of cells that is expected to have the greatest activity and add 5-50 μL to a pilot Eppendorf containing 700 μL of Master Mix and 50 μL of chloroform
 * Wait for the pilot tube to turn yellow
 * Based on this information you can recalculate how many μL of cells to add and how long to let the assay go
 * Remember that the cells have still been growing (for about 30 min or more) while you have been doing the pilot test
 * Add 700 μL of Master Mix to each Eppendorf to be used in the assay.
 * Add 50 μL of chloroform to each Eppendorf.
 * Add 5-50 μL of cells to each Eppendorf and vortex thoroughly
 * The amount added should be based on the results of the pilot test
 * When adding cells make sure to insert the pipette tip into the assay solution of Master Mix and chloroform
 * Count the seconds (10-15 seconds, usually) between each addition of cells to assay
 * The assay begins when the cells are added and needs to be stopped in the same order and timing to ensure accurate results
 * Incubate the assay at room temperature (to slow down fast reactions) or at 37°C (preferred reaction temperature) for between 15 minutes and a few hours (temperature and time are reaction specific and should be based on the pilot test)
 * Stop the reaction with 700 μL stop solution and vortex briefly
 * Be sure to add stop solution in the same order the cells were added and with the same count in between each addition of stop solution as addition of cells (10-15 seconds)
 * The assay stops when stop solution is added
 * Note the amount of time the assay ran
 * Centrifuge the assay, and obtain the OD405 of the supernatant
 * Obtain the OD600 of the sub-cultures.
 * Calculate Miller Units

Master Mix
Make fresh the same day as the sub-cultures and store on ice: Vortex until all the substrate is dissolved&mdash;this can take several minutes Note: Make this mix in a factor greater than 20X due to the small amounts of substrate in the mix.
 * 700 μL Basal Buffer
 * 0.6 mg substrate
 * o-NPG for β-Gal
 * p-NPG for GUS
 * 0.25 μL 20% SDS
 * 2 μL β-Mercaptoethanol (BME; in fume hood)

Basal Buffer (500 mL)
Autoclave
 * 500 mL dH2O
 * 4.3 g Na2HPO4
 * 2.4 g NaH2PO4
 * 0.75 g KCl
 * pH to 7.0 with ~1.5 mL 2N KOH
 * Add 1 mL of 1 M MgSO4·7H2O

1 M Stop Buffer (500 mL)
Autoclave (a precipitate will form)
 * 500 mL dH2O
 * 53 g sodium carbonate (Na2CO3)

Miller Units
mu = [1000 / [T (min) * Vol (mL)]] * [OD405 / OD600]
 * T = minutes assay ran
 * Vol = volume of cells added (5–50 μL)