1. Day 9 (morning): Start the culture for enzymatic measurement.
- Re-suspend culture and transfer 125 μL using a multichannel pipette on a spectrophotometer plate (measure OD600).
NOTE: Caution: Do not dispense the last drop to avoid bubbles, if the OD600 is below 0.3 - 0.6, incubate the remaining cells for 1 - 2 h more based on the reading. The 125 μL culture used at this step can be discarded or transferred back to the original deep well block at the user's discretion.
- Centrifuge the remaining cells in the deep well block for 10 min at 3000 x g and 21 °C. Remove supernatant by inverting.
- Add 200 μL of Z-buffer and vortex.
NOTE: Please see the recipe for Z-buffer in Table 1.
- Centrifuge for 5 min at 3000 x g and 21 °C. Remove supernatant by inverting.
- Add 20 μL Z-buffer and vortex.
- Cover the plate with sealing foil that can resist freeze-thaw cycles. Perform 4 cycles of freeze/thaw using liquid nitrogen in a fume hood and then 42 ºC in a water bath (2 min each).
- Add 200 μL Z-buffer/beta-mercaptoethanol (β-ME)/Ortho-nitrophenyl-β-galactoside (ONPG) (12 mL, 21 μL, 8.4 mg respectively will yield 20 mL solution; always prepare fresh).
NOTE: ONPG will take some time to dissolve properly so prepare the solution an hour before reaching this step. ONPG serves as the substrate for the measurement of β-galactosidase activity.
- Incubate for up to 17 - 24 h at 30 °C (check in between, if color develops earlier proceed to the next step).
2. Day 10 (morning): Stop the reaction and measure.
- Add 110 μL 1M Na2CO3 to stop the reaction and record the time.
- Vortex and centrifuge for 10 min at 3000 x g and 21 °C.
- Take 125 μL of supernatant using multichannel and measure OD420.
NOTE: Caution, do not dispense the last drop to avoid bubbles.
Table 1: The recipe for solutions used in the protocol. The table provides the recipe for the stock and working solutions of the major buffers used in the assay.
| TE buffer 10X (10ml) | |
| Desirable | From the stock |
| 1000mM Tris-Cl (pH 8.0) | 1mL of Tris 1M |
| 10mM EDTA (pH 8.0) | 200uL of EDTA 0.5M |
| Make up the volume with water | |
| TE/LiAc (10ml) | |
| Desirable | From the stock |
| 1X | 1mL TE 10X |
| 0.1M LiAc | 1mL LiAc 1M |
| Make up the volume with water | |
| TE/LiAc/PEG (50 mL) 50% | |
| Desirable | From the stock |
| 1X | 5 mL TE 10X |
| 0.1M LiAc | 5 mL LiAc 1M |
| 40 mL PEG3350 | |
| Z-buffer (1L) pH 7.0 | |
| Na2HPO4 16.1g of heptahydrated or 8.52g of anhydrous |
| NaH2PO4 5.5g of hydrated or 4g of anhydrous |
| KCl 0.75g |
| MgSO4 0.246g of hydrated or 0.12g of anhydrous |
| Maintain pH with HCl |
| All solutions are sterlized before use. |
| Media and agar plates used in the protocol (YPDA, SD-Ura; SD-Trp-Ura and SD-Trp-Ura agar plates) follow the same recipe as in Y1H |
