Sepharose spin column chromatography. A fast, nontoxic replacement for phenol:chloroform extraction/ethanol precipitation.

DNA is often subjected to multiple enzymatic treatments during subcloning procedures. For example, converting a restriction site to another by insertion of a synthetic linker requires five or six enzymatic steps: restriction enzyme digestion, treatment with DNA polymerase (if ends are not blunt), phosphorylation of linkers, ligation of linkers to blunt ends, removal of excess linkers by restriction enzyme digestion, and ligation to circularize the plasmid before transformation into bacteria. Some sequential enzymatic treatments may be performed in a single buffer and do not require an intermediate DNA purification step (1). However, when buffers are incompatible, or if enzymes must be removed, DNA purification by phenol:chloroform extraction followed by ethanol precipitation is a time-consuming (30-60 min), yet often recommended procedure (1). By replacing most extraction and precipitation steps with a 5-min gel filtration spin chromatography procedure, it is feasible to perform multistep procedures in a single day. This procedure removes small molecules (e.g., salt, SDS, and nucleotides) and removes or inactivates most enzymes. DNA is recovered in TE (10 mM Tris, 1 mM EDTA) at the same concentration as input DNA. Although premade columns that effect similar purifications are available commercially, column sizes are fixed so they are less versatile than those made in the lab, and they are not as economical. We use Sepharose CL-6B (Pharmacia, Piscataway, NJ) spin columns for the following applications: