Small Scale Ribosome Prep (via Lindahl Lab)
Grow Cells—DAY 1
1. Inoculate 5ml LB with appropriate antibiotics and grow ON
2. In AM, inoculate 200ml LB with appropriate antibiotics to OD450=0.05, grow
cells to OD450=1.5-2.0. Take OD450 readings every 30min and plot on log paper
3. At OD450=1.4-2.0 harvest cells in a 250ml centrifuge bottle and spin at 8K rpm,
10min.
4. Resuspend pellet dry, add 1ml Buffer A; resuspend well and transfer to 2ml
centrifuge tube.
5. Spin @10K rpm for 5min, decant, resuspend in 1ml Buffer A
6. Split into 2 tubes and add 100ul 15mg/ml lysozyme (fresh) to each tube, mix,
incubate on ice 3min, flash freeze at -80C.
Mini prep—DAY 2
Prepare lysates –
1. Remove tubes from -80 and slow thaw in ice-water bath (take ~2hours)
2. Clarify lysate by spinning in Beckman Optima Ultracentrifuge in MLA 130 rotor
at 22K rpm for 30min using open top 11x34 mm tubes (#343778) either use
immediately or transfer supernatant to 2ml tube flash freeze and store at -80C (reclarify by spinning at 13K rpm for 5min)
Pellet ribosomes – DAY 3
1. Transfer clarified lysate to 2ml sealable tube for MLA 130, top off with Buffer A
as needed and seal. Place in rotor with spacers
2. Spin at 40K rpm for 4 hours, resuspend in 100ul Buffer A, rock overnight at 4C.
3. In AM, transfer to 1.5ml tube, spin at 13K 10min at 4C. Transfer to new tube and
measure OD260 (1:100 dilution).
4. Store ribosomes at -80C.
SOLUTIONS:
Buffer A (autoclave)
HEPES-KOH pH 7.5
MgCl2
NH4Cl
Mercaptoethanol
working con
20mM
60mM
30mM
6mM
Stock
2M
1M
solid
1L 30X
300ml
180ml
48.2g
To make 100ml 1X Buffer A:
3.3ml 30X Buffer A qs to 100ml with water, add 27ul mercaptoethanol store at 4C
HEPES Salt Wash Buffer
HEPES
MgCl2
NH4Cl
Stock
2M
1M
solid
100ml
1ml
3ml
5.4g
MET
d2H20
pure
40ul
to 100ml