Team:Peking/Notebook/Protocols/PCIEG
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<font size=5><font color=#484848><font face="Franklin Gothic Demi Cond"> Protocol by PKU 2010</font></font></font> | <font size=5><font color=#484848><font face="Franklin Gothic Demi Cond"> Protocol by PKU 2010</font></font></font> | ||
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- | [[Team:Peking/Notebook|Notebook]] > [[Team:Peking/Notebook/Protocols|Protocols]] > [[Team:Peking/Notebook/Protocols/PCIEG|Protocol for | + | [[Team:Peking/Notebook|Notebook]] > [[Team:Peking/Notebook/Protocols|Protocols]] > [[Team:Peking/Notebook/Protocols/PCIEG|Protocol for chemical inducible expression of GFP]]<html> |
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- | =Protocol for | + | =Protocol for Chemical Inducible Expression of GFP= |
==Materials:== | ==Materials:== | ||
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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; |
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+ | * Phosphate Buffered Solution (PBS). | ||
==Procedure:== | ==Procedure:== | ||
- | 1. | + | 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. | |
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- | + | 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. | |
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+ | ==Notes:== | ||
+ | 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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Latest revision as of 17:11, 12 October 2010
Contents |
Protocol for Chemical Inducible Expression of GFP
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.