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Yeast Polysome Profiles Adapted from Baim et al., 1985, Mol. Cell. Biol. 5(8):1839-46.
 

Summary: 75
Yeast Polysome Profiles
Adapted from Baim et al., 1985, Mol. Cell. Biol. 5(8):1839-46.
500 ml:
TSM Lysis Buffer: 10 mM Tris-Cl, pH 7.4 0.79 g
100 mM NaCl 2.92 g
30 mM MgCl2-6H2O 3.05 g
For 2 samples, add fresh to 130 ml of above: 1.3 ml of 5 mg/ml cyclohexamide (Sigma C7698)
(Make fresh cycloheximide and heparin 2.6 ml of 10 mg/ml heparin (Sigma H3393)
stock solutions each week. Store at 4C.) 26 l DEPC
100 ml:
2X Gradient Buffer: 15 mM Tris-Cl, pH 7.4 240 mg
140 mM NH4Cl 750 mg
7.8 mM MgOAc-4H2O 168 mg
15% Sucrose: Dissolve 7.5 g Ultra Pure Sucrose in 25 mls 2X Gradient Buffer. Add ddH2O to 50 ml.
50% Sucrose: Dissolve 25 g Ultra Pure Sucrose in 25 mls 2X Gradient Buffer. Add ddH2O to 50 ml.
Foni-inert: 70% (wt/vol) sucrose, 10% (wt/vol) glycerol, 0.01% bromophenol blue.
Prior to collecting yeast, make sure all solutions are at 4C. Have stock of 5 mg/ml cyclohexamide
thawed and on ice. Have IEC centrifuge and carriers precooled. Pre-cool SW 41 Ti rotor.
Pour 12 ml gradients (15 to 50% sucrose; about 5-6 minutes each) in cold room. Use Beckman

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine