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