"
Team:Peking/Notebook/Protocols/MBP
From 2010.igem.org
NOTEBOOK HOME
CountDown
Vistors From...
Protocol by PKU 2010
Notebook > Protocols > Protocol for chemical inducible expression of MBP & sample preparation
Protocol for chemical inducible expression of MBP & sample preparation
Materials:
- HgCl2 store solution with the concentration of 10^-3M
- Overnight bacterial culture or bacterial colonies;
- fuming nitric acid
- IPTG
1. Add 1ml of the overnight bacterial culture or pick a colony to 100ml of LB antibiotic medium, Incubate at 37 degree in a shaker till the OD600 value reaches 0.6.
2. Add 1ml IPTG to the 100ml bacterial to induce the expression of Metal Binding Peptide (MBP) or Lpp-Ompa-MBP or Dsba-MBP.
3. Incubate the culture at 30 degree for 0.5 hour.
4 Supply HgCl2 to the culture to different concentrations of 10^-6M, 10^-7M, 10^-8M and control.
5. Incubate the culture at 30 degree for 40 hours.
6 Harvest cells by spinning at 5000rpm for 5 min. Discard the supernatant, Wash with 6ml of ddH2O for two times.
7 Dehydration the bacteria with freeze dryer, followed by measurement of the weight of cell pellet.
8 Resuspend the pelleted cells in 8ml fuming nitric acid, followed by microwave digestion. Resuspend sample to 25mL. Then sample preparation is completed.
