Experiments
Describe the experiments, research and protocols you used in your iGEM project.
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project
Protocols
Heat shock competent cells
Preparation
Day 1 Inoculate cells in 3.5mL LB medium.
Incubate at 37°C overnight.
Day 2 Measure culture OD at 600nm. Dilute cells in 250mL of LB medium so OD equals to 0.12.
Incubate overnight at 20°C and 180rpm.
Day 3 Measure culture OD at 600nm and dilute to obtain OD600nm=0.6.
Cells must be kept at 4°C during all following steps.
Put on ice for 10min and centrifuge for 10min at 3000rpm and 4°C.
Discard supernatant and resuspend cells in 80mL of fresh TB buffer.
Keep on ice for 10min and centrifuge for 10min at 3000rpm and 4°C.
Discard supernatant again and resuspend cells in 20mL of fresh TB buffer with 7% of DMSO.
Keep on ice for 10min.
Aliquot cells and freeze with liquid nitrogen.
Keep at -80°C.
TB buffer recipe
| HEPES
|
10mM
|
| MnCl2
|
55mM
|
| CaCl2
|
15mM
|
| KCl
|
250mM
|
| KOH
|
|
Dissolve HEPES, CaCl2 and KCl in water. Adjust pH to 6.7 with KOH. Add MnCl2. Filter to sterilize and keep at 4°C.
Tranformation
Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid).
Keep tubes on ice for 30min and heat shock at 42°C for 1min.
Add 500µL of LB medium into each tube and incubate at 37°C for 1h.
Spread cells on Petri dishes in duplicate and incubate at 37°C overnight.
Electro-competent cells
Preparation
Inoculate 15mL of LB with 200µL of an overnight cell culture.
Incubate at 37°C and 180rpm until OD600nm reaches 0.6.
Centrifuge cells for 10 minutes at 4000rpm.
Wash twice with 10mL of 10% glycerol.
Put cells in 200µL of 10% glycerol and use for electroporation.
Transformation=
