Team:UST Beijing/Notebook
PROTOCOLS
Preparing competent cell
1. Incubate E. coli overnight.
2. Dilute cultures in 1:100 and incubate at 37°C and 200rmp for 2-3 hours.
3. Measure Abs600 (between 0.3 and 0.5).
4. Take 3mL samples on ice for 10 min.
5. Centrifuge at 12000rmp for 2 mins and dump the liquid.
6. Add 1mL CaCl2 (0.1mol/L) into samples, resuspend and repeat the last step.
7. Add 500uL CaCl2 (0.1mol/L) into samples, resuspend and repeat the cen-trifugation.
8. Add 100uL CaCl2 (0.1mol/L) into samples, resuspend.
Transformation
1. Take 5uL plasmid into 100uL E. coli BL21 competent cell and put it on ice for 30 min.
2. Heat shock at 42°C for 45s.
3. Add 450uL LB medium into samples incubate at 37°C and 200rmp for 45 min.
4. Take 100uL samples on LB agar medium (contain chloramphenicol) and in-cubate at 37°C.
Veryfication
1. Pick the monoclonal bacteria to 2ml liquid LB medium (contain chloram-phenicol) and culture overnight at 37°C and 220rmp.
2. Transfer 20uL of sample to M9 medium for 4-6 hours at 37°C and 220rmp.
3. Add 100ul samples and blank control (E. coli BL21 without plasmid) into the well of 96-well plates.
4. Add three different doses of PNPG(5，10，20μL) in a 96-well plate by mi-cropipette.
5. Add ddH2O to make sure each well has 200uL in total.
6. Use ultraviolet spectrophotometer to measure Abs405 value every other hour.
Hydrolyzing Ginsenoside
1. Add engineered strain 2mL and M9 medium 200mL (contain Chloram-phenicol) into fermentation tank.
2. Set heating equipment (household appliances which is used to make yo-gurt or wine), choose “yogurt” option.
3. Pump in sterile air and let bacteria grow 4-6 hours.
4. Add ginsenoside powder into fermentation tank directly.
Thin—layer chromatography
1. Prepare Developing solvents 5 ml （CHCl3:CH3OH:H2O=10:2.5:0.5）.
2. Prepare color developing reagent（10%H2SO4） 100mL.
3. Prepare standard sample.
4. Use water saturated n-butyl alcohol to extract hydrolysate.
5. Use capillary tube to point samples onto silicon-glass plate.
6. Put plate into developing solvents, and take out plate when the solvents ar-rive end line.
7. After drying, put plate into color developing reagent and take out immedi-ately.
8. Use hair dryer to accelerate the process of desiccation.