Protocol

Preparation of Plasmid DNA: Toothpick Minipreparation

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks.

MATERIALS

Antibiotic for plasmid selection

Bromophenol blue solution, 0.4% (w/v)

Cresol red solution (10 mM)

EDTA (0.5 M, pH 8.0)

Ethidium bromide (10 mg/ml)

KCl (4 M)

NSS solution

Rich broth

Rich broth agar plates

SYBR Gold

METHOD

1. Grow bacterial colonies, transformed with recombinant plasmid, on rich agar medium (LB, YT, or SOB) containing the appropriate antibiotic until they are approx. 2-3 mm in diameter (approx. 18-24 hours at 37°C . . . [Full Text of this Article]