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Protocol

Double Digestion |Gel Extraction|Miniprep | Chemical Inducible Expression of GFP |DNA Purification |Ligation of Insert DNA | PCR |Preparation of Competent Cells| Site-directed Mutagenesis| Transformation|

Protocol for Chemical Inducible Expression of GFP

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Materials:

• 4 groups of induce solution with a concentration gradient of 10^-7, 10^-5, 10^-3, 10^-2;

• Overnight bacterial culture or bacterial colonies;

• Phosphate Buffered Solution (PBS).

Procedure:

1. Add 20 μl of the overnight bacteria l culture or pick a colony to 5ml of LB antibiotic medium, Incubate at 37 degree in a shaker till the OD600 value reaches 0.4-0.6.

2. Add 0.5 mL of the fresh bacteria l culture and appropriate volume of inducer solution to prepare induction system with the concentration gradient of 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4, 10^-3, 10^-2.

3. Place the induction system at 37 degree for 2 hours.

4. Pellet bacteria l cells by 4 min centrifugation at 4000 rpm, discard the supernatant.

5. Resuspend the pelleted cells in 500 μl of PBS.

6. Transfer 100 uL of bacteria l resuspention into each well of 96-well plate to test the expression of GFP by flow cytometry or Microplate Reader.

Notes:

If desired, time sequential expression of GFP can also be tested, through verifying the incubating time of induction system at 37 degree.