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| - | ß-galactosidase assays were performed as follows. Cells were grown overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM. Overnight cultures were back-diluted 1/100 (50 µL into 5mL) and grown for 3 hours at 37˚ and 225 RPM. OD600 values were taken on the spectrophotometer. Cells were used at OD values between 0.09 and 0.20. 20 µL of our cell culture and 380 µL permeabilization solution (100mM Na2HPO4, 20mM KCl, 2 mM MgSO4, 0.6 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, and 5.4 ul/mL ß-mercaptoethanol) were mixed and placed at 37˚C for 10 minutes. 100 µL of lysed cells were then mixed with substrate solution (60 mM Na2PO4, 40 mM NaH2PO4, 1 mg/mL ONPG, and 5.4 µL/mL ß-mercaptoethanol). When a faint yellow color was observed, 300ul 1M sodium carbonate was added to stop the solution. Miller units were calculated using the following equation:
| + | # Grow cells overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM. |
| - | 1000 (ABS420 / (0.02 mL * t * ABS600). | + | # In the morning, back-dilute overnight cultures 1/100 (50 µL into 5mL) and grow for 3 hours at 37˚ and 225 RPM. The OD should be between 0.09 and 0.20 for 200 µl in a 96-well plate measured in a plate reader (Spectramax M2). |
| - | | + | # Combine 20 µL of cell culture and 380 µL permeabilization solution, mix, and place at 37˚C for 10 minutes. |
| | + | #* Permeabilization Solution: 100mM Na<sub>2</sub>HPO<sub>4</sub>, 20mM KCl, 2 mM MgSO<sub>4</sub>, 0.6 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, and 5.4 µl/mL ß-mercaptoethanol. |
| | + | # Combine 100 µL of lysed cells with 600 µL substrate solution. |
| | + | #* Substrate Solution: 60 mM Na2PO4, 40 mM NaH<sub>2</sub>HPO<sub>4</sub>, 1 mg/mL ONPG, and 5.4 µL/mL ß-mercaptoethanol. |
| | + | # When a faint yellow color is observed, add 300 µl 1M sodium carbonate to stop the reaction. |
| | + | # Miller units are calculated using the following equation: MU = 1000 (ABS<sub>420</sub> / (0.02 * t * ABS<sub>600</sub>)). |
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Latest revision as of 00:43, 30 October 2008
iGEM 2008
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LacZ Assay
- Grow cells overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM.
- In the morning, back-dilute overnight cultures 1/100 (50 µL into 5mL) and grow for 3 hours at 37˚ and 225 RPM. The OD should be between 0.09 and 0.20 for 200 µl in a 96-well plate measured in a plate reader (Spectramax M2).
- Combine 20 µL of cell culture and 380 µL permeabilization solution, mix, and place at 37˚C for 10 minutes.
- Permeabilization Solution: 100mM Na2HPO4, 20mM KCl, 2 mM MgSO4, 0.6 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, and 5.4 µl/mL ß-mercaptoethanol.
- Combine 100 µL of lysed cells with 600 µL substrate solution.
- Substrate Solution: 60 mM Na2PO4, 40 mM NaH2HPO4, 1 mg/mL ONPG, and 5.4 µL/mL ß-mercaptoethanol.
- When a faint yellow color is observed, add 300 µl 1M sodium carbonate to stop the reaction.
- Miller units are calculated using the following equation: MU = 1000 (ABS420 / (0.02 * t * ABS600)).
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