Swartz:Protocols/Ribosome purification: Difference between revisions
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m (New page: See https://docs.google.com/document/d/1NIr0ldJ3qRHlnsK1kKO-WOEMJor2i8oIsP89cbREMxE/edit =Ribosome Purification= Buffers: BME should be added just before use -it may be helpful to make ...) |
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Buffers: | Buffers: | ||
Buffer A | |||
:20 mM Tris HCL pH 7.2 | |||
:100 mM NH_4Cl | |||
:10 mM MgCl_2 | |||
:0.5 mM EDTA | |||
:6 mM BME | |||
Buffer B: | |||
:20 mM Tris HCL pH 7.2 | |||
:100 mM NH_4Cl | |||
:10 mM MgCl_2 | |||
:0.5 mM EDTA | |||
:6 mM BME | |||
:37.7% Sucrose | |||
Buffer C: | |||
:10 mM Tris OAc pH 7.5 | |||
:60 mM NH_4Cl | |||
:5mM Mg(OAc)_2 | |||
:0.5 mM EDTA | |||
:6 mM BME | |||
BME should be added just before use | BME should be added just before use |
Latest revision as of 13:36, 24 July 2012
See https://docs.google.com/document/d/1NIr0ldJ3qRHlnsK1kKO-WOEMJor2i8oIsP89cbREMxE/edit
Ribosome Purification
Buffers: Buffer A
- 20 mM Tris HCL pH 7.2
- 100 mM NH_4Cl
- 10 mM MgCl_2
- 0.5 mM EDTA
- 6 mM BME
Buffer B:
- 20 mM Tris HCL pH 7.2
- 100 mM NH_4Cl
- 10 mM MgCl_2
- 0.5 mM EDTA
- 6 mM BME
- 37.7% Sucrose
Buffer C:
- 10 mM Tris OAc pH 7.5
- 60 mM NH_4Cl
- 5mM Mg(OAc)_2
- 0.5 mM EDTA
- 6 mM BME
BME should be added just before use
-it may be helpful to make 5x stocks of each buffer without BME (and sucrose for Buffer B) and prepare 1x buffers just before use
Day 1:
- Resuspend frozen cells in Buffer A at 1g/mL
- -typically use 10g cells in 10mL Buffer A
- -cells should be from an E. coli fermentation, preferably harvested while still in exponential phase
- -the hand homogenizer can be used for resuspension
- Add ~150U of RNAse inhibitor (from Invitrogen, usually at 10-30U/uL)
- Lyse by flowing through homogenizer 1 pass at >20,000psi
- Spin down lysate at max speed (about 12,000 rpm) for 15min in Sorval RC5B centrifuge in 30mL centrifuge tube
- Pour supernatant into fresh 30mL tube
- -add 2-3mL Buffer A to pellet and add this to fresh tube as well to remove soft top layer
- Spin again for 15min at ~12,000rpm
- Collect supernatant by pouring into fresh Falcon tube
- During spins, prepare Buffer B
- Add 3mL Buffer B to 8 different Type 65 rotor ultracentrifuge tubes
- Pipette ~2-2.5mL of lysate supernatant to each ultracentrifuge tube being careful not to mix
- -there should be visible separation between the 2 solutions
- Mass tubes pairwise to <5mg
- Spin in Beckman L8 80M ultracentrifuge with Type 65 rotor at 4°C, 50,000rpm, 20 hours
- -it is recommended to pre-cool the rotor to 4°C before use
Day 2:
- When ultracentrifugation is complete, there should be a brown ring about halfway up each tube
- Carefully pour off supernatants
- -ribosomes should form a clear, soft pellet on the bottom of the tubes
- Invert tubes and place at 4°C to dry for about 10min being careful not to lose pellets
- Add 250uL Buffer C to each tube
- Rock samples at 4°C for 3-4 hours to resuspend
- -cover tubes with parafilm
Determining ribosome concentration:
- Measure A280 and A260 using spectrophotometer
- -Dilute samples approximately 1000x for measurement
- -A260/A280 should be just under 2
- To calculate concentrations, assume 15 A260 units = 1mg/mL of ribosomes
- -E. coli ribosome MW is 2.7 million Daltons