• Turn on water bath to 42C
• Get Bucket of Ice
• Get DNA Plasmids
• Thaw the competent cells on ice
• Bring bucket of ice to the -80C for this part
• Put overnight tubes on ice (# overnight tubes = # transformations)

Preparing DNA plasmids

• From iGEM wells
1. Add 10uL of Nuclease-free water into well
2. Pipette up and down several times to mix
3. Let sit for a few minutes
• From Amp Resistance stocks
1. Aliquot 1uL of DNA from stock tube into a different microcentrifuge tube
2. Put DNA stock tube back into freezer
3. Add 99uL of sterile water into microcentrifuge tube (concentration 1:100)
4. Aliquot 1uL of the new mix into a second microcentrifuge tube
5. Add 9uL of sterile water into second microcentrifuge tube (concentration 1ng/ul)

Overnight Tubes (start from here if using hydrated DNA or ligations)

1. Take 25uL aliquots of cells into the overnight tubes on ice
2. 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
3. Let sit for 30 minutes on ice
4. Heat Shock the Tubes in the Water Bath by placing the tubes in the water bath (42C) for exactly 40 seconds
5. Place immediately back on ice for 2 minutes
6. Add 200uL of SOB to each tube
7. Place into shaker (200rpm @ 37C) for 1 hour.

Plating

1. Spread cells onto plates
2. Incubate overnight in the incubator (37C)