Team:CSU CHINA/Experiments
Experiments
Chemical transformation
Thaw 50µL DH10B competent E. coli cells on ice.Add 5 µl DNA from a ligation reaction mix or 10-100ng DNA of a plasmid.
Place the mixture on ice for 20-30 minutes.
Heat shock at 42°C for 90 seconds.
Place on ice for 5 minutes.
Pipette 200 µl of room temperature LB media into the mixture.
Incubate at 37°C and 220 rpm for 60 minutes.
Add 50-100 µl of the transformed cells to the selection plate.
Growing overnight cultures
Overnight cultures were prepared under sterile conditions using a Bunsen burnerAdd 3 mL liquid LB media into test tubes
Add 3 μL of appropriate antibiotic（1000x） into the broth
Using the pipette tips, pick a single colony and inoculate the cultures by dipping the tip into the LB broth or by adding 50 μL stored cells
Put caps on the tubes and incubate overnight at 37°C shaking at 200-250 rpm
PCR From Plasmid DNA Template
In a PCR tube on ice, combine 1-10 ng of plasmid DNA or just 1 µL template, 2 µL of 10 µM forward primer, 2 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phanta Mastermix, and sterile water up to 50 µL.
Gently mix the reaction
If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling
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