Griffitts:ß-Gal/GUS assay

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Contents

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:

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 carbonate (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)
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