Team:Manchester/Experiments
Experiments
Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.
Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project
Inspiration
Reagants:
LB broth (Luria Bertrani medium = rich media to grow bacteria)
TSS buffer (to prepare chemically competent cells)
S.O.C. medium (helps obtain the maximal transformation efficiency)
LB agar (gel where bacteria can grow)
Chloramphenicol (CAL) at stock concentration 25mg/ml
Preparation of chemical competent cells:
- Inoculate DH5α cells into 50mL LB and incubate at 37°C
- Monitor growth every 30mins by measuring optical density at 600nm (OD600); until reach OD600 = 0.4-0.6
- Harvest cells and prepare using TSS protocol (see: TSS Protocol)
- Aliquot 200uL and flash freeze at -80°C
LB Agar plates preparation:
- Inoculate DH5α cells into 50mL LB and incubate at 37°C
- Monitor growth every 30mins by measuring optical density at 600nm (OD600); until reach OD600 = 0.4-0.6
- Harvest cells and prepare using TSS protocol (see: TSS Protocol)
- Aliquot 200uL and flash freeze at -80°C
Materials:
5 ml LB broth
5 μl antibiotic
Loops
12 ml culture tube
Methods:
Overnight cultures were prepared under sterile conditions using a Bunsen burner
- Add 5 ml liquid LB media into 12 ml culture tubes
- Add 5 μl of appropriate antibiotic into the broth
- Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth
- Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm
