Swartz:Protocols/1L extract prep

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1L shake flask extract prep:

S30 buffer:

Cell prep:

  • Add single colony or glycerol stock to 5ml defined media
  • Shake at 37C for about 15 hours overnight
  • Autoclave 100mL and 1L flasks
  • Add entire contents of overnight cultures to 100mL starter cultures and shake at 37C
  • Grow to an OD of ~2 and add 50mL to 1L flask
    • -growth usually takes around 4 hours
  • Grow to an OD of ~4-8
    • -stop growth during exponential phase
    • -stop growth before pH drops below 6
  • Spin down cells and add to a 50mL Falcon tube
    • -resuspend with S30 buffer to ~45mL and spin down
    • -repeat S30 wash
  • Measure mass, and store at -80C (or proceed to extract prep)

Extract prep:

  • Thaw pellets in 1mL S30 buffer per g cells
  • Prepare cell homogenizer by washing with water, NaOH, and EtOH as per instructions on machine
  • Resuspend pellets using hand homogenizer
  • Lyse by flowing through homogenizer 1 pass at >20,000psi
  • (After the initial resuspended pellet has gone through the homogenizer, I usually lower the pressure on the valve to recover the rest of the holdup volume, which is significant for these size volumes.)
  • Spin down in 50ml tubes for 30min at 13,000rpm
    • -repeat spin
  • Incubate for 80min at 37C in the dark