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Team:Northwestern/makecells
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Making Competent Cells
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
- 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
