Combined protocol for selection and isolation of recombinant plasmids using multiwell plates.

Ligation of a DNA restriction fragment into a plasmid vector, followed by isolation of a recombinant plasmid, is a fundamental procedure in molecular biology. Standard protocols require at least two days to go from unligated DNA to the isolation of sufficient quantities of recombinant plasmid for restriction enzyme analysis or DNA sequencing (1,3). The two most timeconsuming steps involve the selective overnight growth of bacterial colonies on antibiotic agar plates and the subsequent growth of isolated colonies in liquid cultures, which usually requires at least six hours. I have developed a protocol that combines these two steps by selecting serially diluted bacteria in liquid cultures grown in 48-well tissueculture dishes. Here I describe how this procedure makes it possible to complete the entire process from ligation to the isolation of recombinant plasmid DNA in 24 h. Ten micrograms of the 15-kb plasmid pA8-1, which contains a portion of the gene encoding the murine transcription factor αA-CRYBP1 (2), and 0.25 μg of the 2.9-kb vector pBluescript® SK(-) (Stratagene, La Jolla, CA, USA) were digested separately for 30 min at 37°C with 20 units and 10 units, respectively, of the restriction enzyme XbaI. Twelve units of calf intestinal alkaline phosphatase were then added to the pBluescript digestion, and both reactions were incubated for an additional 15 min. After electrophoresis of the digested plasmids in a 1.0% TAE-buffered agarose gel, a 2.4-kb and a 1.9-kb fragment of pA8-1 and the pBluescript bands, respectively, were excised from the gel (Figure 1). The three gel slices were placed together into a single 1.5-mL microcentrifuge tube, and the DNA was extracted using the Geneclean® II DNA purification kit (Bio 101, La Jolla, CA, USA), yielding a final volume of 20 μL. Ten microliters of the co-purified vector and insert DNA were added to a 20-μL reaction containing 1.0 mM rATP, 1× ligation buffer (Stratagene) and 7.5 units of T4 DNA ligase (Stratagene). After 4 h at room temperature, 10 μL of the ligation reaction were used to transform 100 μL of XL1-Blue Supercompetent E. coli cells (Stratagene) according to the supplier’s instructions. The bacteria plus DNA were added to 900 μL of S.O.C. medium (3) and placed in a shaking 37°C incubator for 1 h. At this point in a standard protocol, a portion of the bacterial solution would be spread onto ampicillin plates to initiate the overnight growth of colonies. If the cells are plated at a reasonably low density, each of these colonies should be clonally derived from a single bacterium and, thus, possess only a single type of plasmid. The protocol described here is based on the hypothesis that serial dilutions of the bacteria in liquid medium containing ampicillin should also yield a concentration of cells at which all of the bacteria in a certain volume of medium contain identical plasmids. Thus, 100 μL of the above bacterial solution were diluted into 25 mL of LB medium containing ampicillin (50 μg/mL). One