Swartz:Protocols/1L extract prep

https://docs.google.com/document/d/1KdksMWFTIV8TRWiV7-AOcve_uCsxq2f6B5__DB-s9fI/edit

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