Swartz:Protocols/1L extract prep: Difference between revisions
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*Incubate for 80min at 37C in the dark | *Incubate for 80min at 37C in the dark | ||
[[Category:Swartz_Protocols]] | [[Category:Swartz_Protocols]][[Category:Swartz_Protocols]][[Category:Cell_Free]] |
Latest revision as of 16:54, 28 May 2012
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