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
Materials:
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 (see: TSS Competent E. coli Protocol):
- Inoculate DH5α cells into 50mL LB and incubate at 37°C
- Monitor growth every 30mins by measuring optical density at 600nm (OD600) until it reaches OD600 = 0.4-0.6
- Harvest cells and prepare using TSS protocol (see: TSS Competent E. coli Protocol)
- Aliquot 200μL and flash freeze at -80°C
LB Agar plates preparation:
- Prepare LB containing chloramphenicol (CAL) (at 25μg/ml)
- Melt LB in microwave (defrost setting for 15mins)
- Cool LB by running cold water over
- Stock of 25mg/ml CAL → so add 400μl CAL to 400ml LB
- Pour plates (in fume hood) and allow to solidify
Chemical Transformation:
- Add 1μl of DNA to 50μl of competent cells, mix well and place on ice for at least 30mins
- Heat shock cells at 42°C for 30secs, followed by 2min incubation on ice
- Add 450μl of SOC medium tot he cells and incubate for 45min at 37°C (to allow (antibiotic resistance) protein expression)
- Plate and spread (glass spreader sterilised over a flame and in ethanol) 50, 100, and 200μl of the cells into the agar plates made previously
- Incubate at 37°C for 2h
Materials:
Dimethyl sulfoxide (DMSO)
Polyethylene glycol (PEG)
MgCl2 stock solution
LB or SOC liquid medium
Ice
Methods:
- Inoculate DH5α E. coli cells into 50ml LB broth and incubate at 37°C
- Prepare the TSS buffer while waiting for the culture to grow and place it on ice (see: Table below)
- Monitor growth of the culture every 30min by measuring the optical density at 600nm wavelength (OD600) until it reaches OD600 = 0.4-0.6 (takes approximately 2-3h)
- Once the proper optical density has been achieved, take 100mL of culture and centrifuge under 2.700xg for 10 min at 4°C
- Resuspend each tube in 5ml of pre-chilled TSS buffer with gentle vortexing
- Chill TSS suspended cells on ice for 15 min
- Aliquot 200μL of TSS suspended cells while ensuring the cells remain well mixed
- Cells can be used immediately or stored at -80°C
TSS Buffer Composition
| Component | Stock (M) | Amount |
|---|---|---|
| MgCl2 | 2 | 0.300ml |
| DMSO | - | 1ml |
| PEG (3350 or 8000) | - | 2g |
| LB Medium | to final volume of 20ml | tofinal volume of 20ml |
