IGEM:Harvard/2010/General Protocols

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.

DNA Digests

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: 1.0 uL
  • Enzyme 2: 1.0 uL
  • Buffer: 2.0 uL
  • Water: ____ uL

____________________________________

• • Total: 20.0 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.