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

Solutions

Master Mix

Make fresh the same day as the sub-cultures and store on ice:

• 700 μL Basal Buffer
• 0.6 mg substrate
  • o-NPG for β-Gal
  • p-NPG for GUS

Vortex until all the substrate is dissolved—this can take several minutes

• 0.25 μL 20% SDS
• 2 μL β-Mercaptoethanol (BME; in fume hood)

Note: Make this mix in a factor greater than 20X due to the small amounts of substrate in the mix.

Basal Buffer (500 mL)

• 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

Autoclave

• Add 1 mL of 1 M MgSO₄·7H₂O

1 M Stop Buffer (500 mL)

• 500 mL dH₂O
• 53 g sodium bicarbonate (Na₂CO₃)

Autoclave (a precipitate will form)

Miller Units

mu = [1000 / [T(min)*Vol(mL)]] * [OD₄₀₅ / OD₆₀₀]

• T = minutes assay ran
• Vol = volume of cells added (5–50 μL)