Team:PKU Beijing/Notebook/Protocol/Chemical Inducible GFP

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{{PKU_Beijing/Sidebar_Notebook}}
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{{PKU_Beijing/Header2}}
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[[Team:PKU_Beijing/Notebook|Notebook]] > [[Team:PKU_Beijing/Notebook/Protocol|Protocol]] > [[Team:PKU_Beijing/Notebook/Protocol/Chemical_Inducible_GFP|Chemical Inducible Expression of GFP]]
==='''Protocol for chemical inducible expression of GFP'''===
==='''Protocol for chemical inducible expression of GFP'''===
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[[Media:PKU_Protocol_for_chemical_inducible_expression_of_GFP.pdf|download PDF version]]
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<html><img src="https://static.igem.org/mediawiki/2009/9/91/PKU_Adobe_Reader_Logo.jpg" width=20></html>[[Media:PKU_Protocol_for_chemical_inducible_expression_of_GFP.pdf|download PDF version]]
'''Materials:'''  
'''Materials:'''  
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*4 groups of induce solution with a concentration gradient of <math>10^{-7}, 10^{-5}, 10^{-3}, 10^{-2}</math>;  
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*4 groups of induce solution with a concentration gradient of 10^-7, 10^-5, 10^-3, 10^-2;  
*Overnight bacterial culture or bacterial colonies;  
*Overnight bacterial culture or bacterial colonies;  
*Phosphate Buffered Solution (PBS).  
*Phosphate Buffered Solution (PBS).  
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'''Procedure:'''<br>
'''Procedure:'''<br>
1. Add 20 μl of the overnight bacterial 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. <br>
1. Add 20 μl of the overnight bacterial 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. <br>
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2. Add 0.5 mL of the fresh bacterial culture and appropriate volume of inducer solution to prepare induction system with the concentration gradient of <math>10^{-9}</math>, <math>10^{-8}, 10^{-7}, 10^{-6}, 10^{-5}, 10^{-4}, 10^{-3}, 10^{-2}</math>. <br>
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2. Add 0.5 mL of the fresh bacterial 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. <br>
3. Place the induction system at 37 degree for 2 hours. <br>
3. Place the induction system at 37 degree for 2 hours. <br>
4. Pellet bacterial cells by 4 min centrifugation at 4000 rpm, discard the supernatant. <br>
4. Pellet bacterial cells by 4 min centrifugation at 4000 rpm, discard the supernatant. <br>

Latest revision as of 10:07, 9 October 2009
 
Notebook > Protocol > Chemical Inducible Expression of GFP

Protocol for chemical inducible expression of GFP

download PDF version

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 bacterial 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 bacterial 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 bacterial 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 bacterial resuspention into each well of 96-well plate to test the expression of GFP by flow cytometry or Microplate Reader.

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

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