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Revision as of 15:07, 16 June 2017
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
Ice
Selection plates
Methods:
- Thaw 50µL competent E. coli cells on ice for 10 minutes
- Add:
- 5-10 µl DNA from a ligation reaction mix or
- 10-100ng DNA of a known plasmid
- Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 950 µl of room temperature SOC or LB media into the mixture.
- Incubate at 37°C and 200-250 rpm for 60 minutes.
- Mix the cells thoroughly by flicking the tube and inverting.
- Spread:
- For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
- Pellet cells at 8000rpm for 3 minutes
- Remove and dispense 600 µL of supernatant
- Re-suspend cells by light vortexing
- Plate resuspended cells as above
- For known plasmid: 10 & 100 µL of each transformation reaction onto a selection plate. For the rest of 890 µL:
- Pellet cells at 8000rpm for 3 minutes
- Remove and dispense 600 µL of supernatant
- Re-suspend cells by light vortexing
- Plate resuspended cells as above
- For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
- Incubate overnight at 37°C with plates upside down.
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
