April 26 1990
Matthew S. Holt
NOTE: The next spin will require the temperature in the RC-5B to be above 15 degrees C so change the temperature setting on the centrifuge to 20 degrees C and leave the door open. You will also need the SM-34 rotor to be above 15 degrees C.
25% sucrose 50 mM Tris*HCL, pH 8.0 1 mM EDTA, pH 8.0
0.1% Triton X-100 50 mM Tris*HCL, pH 8.0 60 mM EDTA, pH 8.0 Shelf life of 3 months
30% polyethelyne glycol (PEG f.wt.8000) 1.5 M NaCl
prepare 2 ml for each sample made fresh each time 3.0 ml 1 M Tris pH 8.0 9.0 ml sterile ddH2O 60 mg Lysozyme 12 ml - enough for 6 samples.
Dissolve 42 grams of CsCl2 in 40 ml sterile 1X TE pH 8.0
Ethidium bromide is a powerful mutagen and moderately toxic. Gloves should be worn when handling. Ethidium bromide waste is contained and disposed of through W.U.M.S. Hazardous Waste Management Office. UV can cause severe burns. Always wear eye protection.
Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp. 1.42-1.46.