Team:Gaston Day School/Notebook
Notebook
On this page, Gaston Day School iGEM team would like to share with you our notes during the experiments from 9/01/2017 to 10/23/2017. The team leader Heena Saqib records the data patiently.
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.
