Protocol

Calcium Phosphate Transfection of 3T3 Fibroblasts

Adapted from Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 2nd edition, by Michael F. Carey, Craig L. Peterson, and Stephen T. Smale. CSHL Press, Cold Spring Harbor, NY, USA, 2008.

INTRODUCTION

Mixing DNA with calcium chloride in a phosphate buffer results in the formation of a DNA/calcium phosphate precipitate which, when layered onto cultured cells, is taken up by endocytosis. The method is used widely for transfecting fibroblasts and other adherent cell types and can be performed easily with reagent-grade chemicals. This protocol describes the calcium phosphate transfection of murine 3T3 fibroblasts. Briefly, cells are grown on 60- or 100-mm tissue culture dishes and passaged 1 d prior to transfection, such that they are ~50% confluent at the time of transfection. This allows the cells to be transfected when most are not in direct contact with other cells. It also allows the cells to reach 100% confluence ~48 h post-transfection, when most transiently transfected cells are harvested. A precipitate containing DNA and calcium phosphate is formed by gradually mixing a phosphate-containing buffer with a solution of DNA and calcium chloride. The mixture is then layered onto a plate containing the adherent cultured cells and growth medium. After the cells have been exposed to the precipitate for a defined period of time, the medium containing the precipitate is removed and fresh medium is added to prevent toxicity. The procedure is suitable for performing both transient and stable transfection experiments.