3 Purification of Nucleic Acids From Eukaryotic Cells
57
DNAses and endonucleases), SDS (to solubilize membranes) and proteinase
K (a protease which destroys most contaminating DNAses and other proteins). This is followed by an extraction with phenol ( Sambracket al. 1989).
Materials
• mammalian cells
• 25:24:1 phenol:chloroform:isoamyl alcohol (with Tris-buffered phenol,
see Subprotocol 1, Materials)
• citric acid
• 0.5 M EDTA (pH 8.0)
• glucose
• phosphate-buffered saline (PBS) or Tris-buffered saline (TBS)
• Proteinase K (20 mg/ml)
• 10mg/ml RNAse (DNAse free)
• 10% SDS
• sodium citrate
• 5 M sodium chloride
• TE-buffer (pH8.0) (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH8.0))
• 1 M Tris-HCl (pH 8.0)
• liquid nitrogen
• sterilized H20
• microcentrifuge tubes
• polypropylene tubes (50 ml)
• Erlenmeyer flask
• microcentrifuge
• cell culture centrifuge
• water bath
• mortar and pestle
• chemical hood
Reagents
Supplies
57
DNAses and endonucleases), SDS (to solubilize membranes) and proteinase
K (a protease which destroys most contaminating DNAses and other proteins). This is followed by an extraction with phenol ( Sambracket al. 1989).
Materials
• mammalian cells
• 25:24:1 phenol:chloroform:isoamyl alcohol (with Tris-buffered phenol,
see Subprotocol 1, Materials)
• citric acid
• 0.5 M EDTA (pH 8.0)
• glucose
• phosphate-buffered saline (PBS) or Tris-buffered saline (TBS)
• Proteinase K (20 mg/ml)
• 10mg/ml RNAse (DNAse free)
• 10% SDS
• sodium citrate
• 5 M sodium chloride
• TE-buffer (pH8.0) (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH8.0))
• 1 M Tris-HCl (pH 8.0)
• liquid nitrogen
• sterilized H20
• microcentrifuge tubes
• polypropylene tubes (50 ml)
• Erlenmeyer flask
• microcentrifuge
• cell culture centrifuge
• water bath
• mortar and pestle
• chemical hood
Reagents
Supplies