Create Needed Solutions

• SOB
• CCMB80 (Ion Solution)

Grow Cells Overnight

Measuring Optical Density

Preparing Solutio

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

Preparing the Spectrophotometer

• Turn on the spectrophotometer
• When ready to measure optical density, blank with regular SOB solution
• Get cuvettes ready

Measuring the Optical Density

• At 2 hours measure the OD
• 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
• If it exceeds .4, discard solution and start over

Centrifuging the Cells

• When the solution has an OD between .35 and .4
• Split into chilled 50mL centrifuge tubes and put on ice for 10 minutes (Have centrifuge tubes chilled in -20C freezer before-hand)
• Centrifuge for 10 minutes (4000rpm @ 4C) (Make sure to have a pellet at the bottom of the tube)
• Decant the supernatant and leave upside down on paper towel for a minute
• 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
• Decant the supernatant and leave upside down on paper towel for a minute
• Resuspend the pellet in 2mL of CCMB80 solution (Add the solution and just shake the tube to resuspend)

Putting the Cells on Ice

• Aliquot the total solution into microcentrifuge tubes (Try to break it up into smaller amounts in many microcentrifuge tubes)
• Place the microcentrifuge tubes into the -80C freezer for later use