Griffitts:ß-Gal/GUS assay: Difference between revisions
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mu = [1000 / [T(min)*Vol(mL)]] * [OD<sub>405</sub> / OD<sub>600</sub>] | mu = [1000 / [T(min)*Vol(mL)]] * [OD<sub>405</sub> / OD<sub>600</sub>] | ||
* T = minutes assay ran | * T = minutes assay ran | ||
* Vol = volume of cells added (5 | * Vol = volume of cells added (5–50 μL) |
Revision as of 10:53, 16 April 2008
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 μL substrate
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)