May 1 1990
Special solutions required:
NOTE: This procedure can be scaled up many fold. For cell culture volumes over 25 mls reprecipitate with isopropanol after step 9 since a great deal of KOAc is still held in large pellets (which lowers pH). Before banding the DNA in CsCl check that the DNA solution is of neutral pH.
stock solution volume final concentration 40% sterile glucose 2.27 ml 50 mM 0.5 M EDTA, pH 8 2.0 ml 10 mM 1M Tris-HCl, pH 8 2.5 ml 25 mM sterile ddH2O 93.23 ml Total: 100.0 ml Use all sterile stock solutions. Store at 4 degrees C.
stock solution volume final concentration 1N NaOH 2.0 ml 0.2 N 10% SDS 1.0 ml 1% sterile ddH2O 7.0 ml Total: 10.0 ml Prepare fresh solution prior to use.
3 M Potassium Acetate:
stock solution volume 5 M KOAc 60 ml glacial acetic acid 11.5 ml ddH2O 28.5 ml 100 ml Filter sterilize. The resulting solution is 3 M potassium and 5 M acetate and has a pH of about 4.8.
RNAase (10 mg/ml):
Dissolve 100 mg RNAase A (pancreatic RNAase) in 10 ml 10mM Tris-HCl 15 mM NaCl. Heat to 100 degrees C (in a beaker of boiling water) for 15 minutes. Cool slowly to room temperature. Dispense into aliquots and store at -20 degrees C.
Sambrook J. Fritsch E.F. and T. Maniatis (1989). Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp.1.25-1.28.