Solutions:

• SOB
• CCMB80 (Ion Solution)
• Magnesium chloride

Grow Cells Overnight

1. Start with Top 10 Cells (from Culture) and Grow
2. Take single colony from plate, and swirl in ~5mL of SOB in overnight tube
3. Grow the Cells in shaker (200rpm @ 37C) for ~16 hours

Preparing Solution

1. Aliquot 100mL of SOB into an Erlenmeyer Flask
2. Add .5mL of 2M magnesium chloride (MgCl2)
3. Autoclave
4. Rinse flask with water for ~1minute to cool
5. Add 1mL of overnight SOB + cell solution
6. Put into shaker (200rpm @ 37C) for 2 hours

Measuring Optical Density (OD)

1. Turn on the spectrophotometer
2. When ready to measure optical density, blank with regular SOB solution
3. Get cuvettes ready
4. At 2 hours measure the OD
5. Continue to measure the OD every 20-30 minutes until it reaches .35
• Note: This growth is exponential, so be quick if close to .35
6. If it exceeds .4, discard solution and start over

Centrifuging the Cells

1. When the solution has an OD between .35 and .4
2. Split into chilled 50mL centrifuge tubes and put on ice for 10 minutes
• Have centrifuge tubes chilled in -20C freezer before-hand
3. Centrifuge for 10 minutes (4000rpm @ 4C)
• Make sure to have a pellet at the bottom of the tube
4. Decant the supernatant and leave upside down on paper towel for a minute
5. Resuspend the pellet in 30mL of CCMB80 solution
• Have the CCMB80 solution ice cold before this
• Gently do this by swishing up and down in transfer pipet
• This may take upwards of 10 minutes, be patient
6. Decant the supernatant and leave upside down on paper towel for a minute
7. Resuspend the pellet in 2mL of CCMB80 solution
8. Add the solution and just shake the tube to resuspend

Putting the Cells on Ice

1. Aliquot the total solution into microcentrifuge tubes
2. Try to break it up into smaller amounts in many microcentrifuge tubes
3. Place the microcentrifuge tubes into the -80C freezer for later use