Preparing & Isolating Ribosomes

Patricia L. Clark
last updated: 12/7/01

PREPARING

• Inoculate 100 ml of minimal media with 2 ml of a fresh O/N culture of Salmonella strain 7136.
• Grow at 30degC until 2x10E8 cells/ml
• Infect with 5-/13- phage to moi=10
• Continue to culture at 30degC for 1.5 hr
• Quickly chill cultures on ice, adding (2) 8 ml R buffer ice cubes to each culture to speed chilling; pour into pre-chilled SS34 or GSA bottles; spin @ 5K for 10 min
• Resuspend pellet in 0.5 ml R+OG buffer, split into two microfuge tubes
• Freeze/store in -85degC; or, freeze cells in liq.N2 and store @ -30degC indefinitely

ISOLATING

• Make 17 ml sucrose gradients: 16 ml linear gradient of 10-30% sucrose (w/w) in R buffer, with 0.75 ml 60% sucrose cushion (plus ~0.25 ml sample)
• Thaw frozen cells in RT H2O bath to initiate lysis (can add 75 ul RNase + 75 ul 0.5 M EDTA to destroy ribosomes, if desired…incubate 10 min at RT for complete destruction)
• Spin in microfuge @ 14 K for 1.5 min
• Layer lysate supernatant onto gradients
• Spin 18 K rpm for 18 hr in SW28.1 rotor, or some other combination of speed/time to give w2/sec=2.3x10E11 (max speed = 28 K rpm, for 7.25 hr)
• Fractionate gradients using peristaltic pump (pumping from bottom, at 1 ml/min) making thirty (30) 0.57 ml fractions
• Check A260 of fractions: make 12.5-fold dilutions (420 ul R buffer + 80 ul fraction) and measure with Baker lab UV spec; good A260 for 50S peak ~ 0.6