Growing a Bacterial E. coli Culture
E. coli is a typical bacteria used for molecular cloning. Long-term storage of BioBrick plasmids is in E. coli suspended in glycerol (500ul cells:500ul glycerol) and kept at -80˚C. Growing E. coli is performed at 37˚C, at which temperature they divide every 20 minutes.
- Day1: Making streaks from glycerol stocks
- Warm agar plate at 37˚C for 10 minutes
- Label lid of plate with what you're streaking on it, and your name, and the date
- Locate desired glycerol stock in -80˚C freezer and, using a sterile toothpick, scrape out a tiny bit and streak on plate
- Incubate plate overnite at 37˚C
- Day2: Growing liquid cultures
- Label 15mL falcon tube and add 5mL of LB media + selective antibiotic
- Using 200uL tip w/o filter, touch streak or single colony and put tip in falcon tube
- Grow culture overnite in a shaking 37˚C incubator
Plasmid Minipreps: Qiagen Miniprep Kit
To extract plasmid DNA from bacteria, perform a miniprep using the Qiagen miniprep protocol.
Use appropriate enzyme to cut the BioBrick plasmids or parts to be ligated, taking care as to whether you're performing a "front-end" or "back-end" ligation.
- Write out exactly what you're building, i.e. part names, sizes, etc
- In a 1.5mL centrifuge tube, set up digest reaction as follows:
- Plasmid DNA ____ uL = ~1000ng
- Enzyme 1 1 uL
- Enzyme 2 1 uL
- Buffer 2 uL
- Water ____ uL
- Total 20 uL
(Note: Always be sure that the TOTAL enzyme concentration is never >10% of TOTAL volume, i.e. 2uL enzymes/20uL total reaction is 10%.)
- Mix tube well and digest at 37˚C for at least 5 minutes. If you are including a phosphotase enzyme, you need to deactivate it by next incubating the tube at 65˚C for 10 minutes.
- Proceed to gel purification.