15 Cloning Vectors
245
Subprotocol 4
"Mini-Prep" Method for Plasmid Isolation
This method is recommended for rapid isolation of plasmids from a high
number of clones (Birnboim 1979, Ish-Horowicz 1981). The isolated DNA is
partially purified and is useful primarily for analytical restriction analysis.
The yield is about 1-5!-lg. If the clones bear a low copy-number plasmid, use
5 ml ofbacterial culture and double the volumes of the corresponding solutions.
Materials
• Lysozyme
• RNase A (1 mg/ml in 5 mM Tris-HCl (pH 8.0), heated 10 min at 70°C)
• LB medium (see Subprotocol 1, Materials)
• Solution A (25 mM Tris-HCl (pH 8.0), 50 mM glucose, 10 mM EDTA, 2
mg/mllysozyme). Add the lysozyme just before use.
• Solution B (0.2 M NaOH, 1% SDS). Make fresh each time.
• Solution C (3M Na-acetate (pH 4.8)): Dissalve 40.8 g sodium-acetate · 3
H 2 0 in a small volume ofH20, adjust the pH with glacial acetic acid and
add water to 100 ml).
• Phenol: Dissalve crystallin phenol carefully at 37°C, add 10 mM Tris, 1
mM EDTA solution (pH 7.5) and store at room temperature. The phenol
will be in the bottarn layer.
• Chloroform/isoamyl alcohol (24/1 v/v)
• 95% ethanol
• 70% ethanol
• Sterile distilled water
• 10 ml tubes with caps
• Sterile toothpicks
• Eppendorf centrifuge
• 1.5 ml tubes for the Eppendorf centrifuge
Reagents
Salutions
Supplies
245
Subprotocol 4
"Mini-Prep" Method for Plasmid Isolation
This method is recommended for rapid isolation of plasmids from a high
number of clones (Birnboim 1979, Ish-Horowicz 1981). The isolated DNA is
partially purified and is useful primarily for analytical restriction analysis.
The yield is about 1-5!-lg. If the clones bear a low copy-number plasmid, use
5 ml ofbacterial culture and double the volumes of the corresponding solutions.
Materials
• Lysozyme
• RNase A (1 mg/ml in 5 mM Tris-HCl (pH 8.0), heated 10 min at 70°C)
• LB medium (see Subprotocol 1, Materials)
• Solution A (25 mM Tris-HCl (pH 8.0), 50 mM glucose, 10 mM EDTA, 2
mg/mllysozyme). Add the lysozyme just before use.
• Solution B (0.2 M NaOH, 1% SDS). Make fresh each time.
• Solution C (3M Na-acetate (pH 4.8)): Dissalve 40.8 g sodium-acetate · 3
H 2 0 in a small volume ofH20, adjust the pH with glacial acetic acid and
add water to 100 ml).
• Phenol: Dissalve crystallin phenol carefully at 37°C, add 10 mM Tris, 1
mM EDTA solution (pH 7.5) and store at room temperature. The phenol
will be in the bottarn layer.
• Chloroform/isoamyl alcohol (24/1 v/v)
• 95% ethanol
• 70% ethanol
• Sterile distilled water
• 10 ml tubes with caps
• Sterile toothpicks
• Eppendorf centrifuge
• 1.5 ml tubes for the Eppendorf centrifuge
Reagents
Salutions
Supplies