Prepare Required Solutions
  • SOB
  • Sterile Water
Prepare Lab for Procedure
  • Turn on water bath to 42C
  • Get Bucket of Ice
  • Get DNA Plasmids
Thaw Required Solutions
  • Thaw the competent cells on ice (Bring bucket of ice to the -80C for this part)
  • Put overnight tubes on ice (# overnight tubes = # transformations)
Prepare DNA
  • From iGEM Wells
    • Add 10uL of Nuclease-free water into well
    • Pipette up and down several times to mix
    • Let sit for a few minutes
  • From AMP Resistance Stocks
    • Aliquot 1uL of DNA from stock tube into a different microcentrifuge tube
    • Put DNA stock tube back into freezer
  • Add 99uL of sterile water into microcentrifuge tube (concentration 1:100)
    • Aliquot 1uL of the new mix into a second microcentrifuge tube
    • Add 9uL of sterile water into second microcentrifuge tube (concentration 1ng/ul)
  • Let DNA sit on ice for 5 minutes after resuspending
    • Tips: When inserting DNA, put DNA into the solution, and leave the tip in it
Prepare Overnight Tubes (start from here if using hydrated DNA)
  • Take 25uL aliquots of cells into the overnight tubes on ice
  • Add 1uL of DNA to each overnight tube on ice
    • Inject the DNA into the solution at the bottom
    • Leave tip in the overnight tube
  • Let sit for 30 minutes on ice
  • Heat Shock the Tubes in the Water Bath
Place the tubes in the water bath (42C) for exactly 40 seconds
  • Place immediately back on ice for 2 minutes
  • Add 200uL of SOB to each tube
  • Place into shaker (200rpm @ 37C) for 1 hour
Preparing Plates
  • Spread cells onto plates
  • Incubate overnight in the incubator (37C)
Making an Overnight Culture
  1. Take 5mL LB and put in a round bottom 5mL tube.
  2. Add 5 μl antibiotic (100x) to the tube.
  3. Take a p200 Tip and lightly touch a colony.
  4. Eject the tip into the culture and put the cap on (not completely).
  5. Grow overnight for 12-16 hours.
  6. Left over overnight cultures (after mini prepping) can be used to make glycerol