Copacabana Method for Spreading E. coli and Yeast Colonies

Molecular biology techniques require the isolation of individual clones for analysis after bacterial or yeast transformation. For E. coli and Saccharomyces cerevisiae, the dispersion of colonies on plates has traditionally involved the use of a glass spreader—a bent glass rod that could be repeatedly flame sterilized and used to spread a small aliquot of microorganisms from the center of the plate to the periphery to cover the entire plate (1,2). For many investigators, learning to disperse bacteria on solid media is one of their first exposures to molecular biology techniques. We describe an alternative method for using sterile glass balls for the dispersion of bacteria and yeast on solid media that requires less technical skill on the part of the operator and avoids smearing colonies or damaging the agar. It also eliminates the need for breakable glass spreaders and open containers of ethanol required for repeated flaming. Glass balls (4 mm) obtained from Thomas Scientific (Swedesboro, NJ, USA) were autoclaved in 100-mL milk dilution bottles. One Shot Top10 Competent cells and the pBADThio TOPO TA Cloning Kit were obtained from Invitrogen (Carlsbad, CA, USA). A PCR product was generated in our laboratory, ligated into the vector, transformed into the bacteria per the manufacturer’s directions, and spread on LB media containing 50 μg/mL carbenicillin. A TRP1-containing yeast expression vector was transformed into the strain Y499 and plated onto tryptophan-deficient complete minimal yeast media using standard methods (3). For E. coli plating, three to five sterile glass balls were shaken onto a 100mm LB/carbenicillin plate after the plate had been warmed in an oven at approximately 40°C with the lid vented to remove condensation. The glass balls were dispensed by flaming the cap and neck of the milk dilution bottle and gently shaking them out of the bottle a few centimeters above the media, three or four per 100-mm plate. E. coli transformation mixture (50–150 μL) was aliquoted onto the center of the media, and the lid was placed back onto the plate. Then, we agitated the plate with a random shaking motion so that the glass balls would roll over the entire surface of the plate. Once the liquid is absorbed and the surface of the media is dry, this process is finished (usually 30–60 s if the plate has been sufficiently pre-warmed). The dry plate is inverted, the beads discarded, and we incubate the media at 37°C overnight. Similar volumes and numbers of beads are used for yeast media for selection by complementation of auxotrophic markers, such as uracil, tryptophan, or leucine-deficient complete minimal media for yeast oneand two-hybrid screens. For either organism, if the plate size is larger, we scale the number of glass balls and volume of transformation mixture proportionately. For example, for a 150-mm plate, we routinely plate 350 μL transformation mixture and use 8–12 glass balls (Table 1). A laboratory strain of E. coli was transformed with the product of a cloning reaction and plated onto the selective media using the Copacabana method. The glass balls are shown in Figure 1A on the surface of a LB/carbenicillin plate before plating. A plate suitable for this purpose allows the balls to move freely with the lid in place; plates with less than 5 mm between the surface of the media and the inner surface of the lid cannot be used. The 100-mm plates we use in our laboBenchmarks