( by UV transillumination on gels les...)Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities

by UV transillumination on gels, less DNA was visible than expected. In an effort to investigate the discrepancy between the absorbance at 260 nm and the amount of DNA visualized, we used quantitative PCR to measure the concentration of the plasmid yielded by each method. The concentrations of pHSVlac as determined by absorbance and by quantitative PCR were similar for methods A, B and D. However, for method C, absorbance apparently overestimates the yield by 2-fold. Presumably a copurified compound accounts for half of the absorbance.

TRANSFECTION EFFICIENCIES
RR1 cells were transfected equally well with pHSVlac prepared by methods A, B and D, but were less efficiently transfected with pHSVlac prepared by method C (see table), whether determining transfection efficiencies by counting blue cells or by measuring b-galactosidase activity. The lower than expected concentration of plasmid from method C may partly account for the low transfection efficiencies. Of the four methods, method A has the advantage of lowest cost, but is particularly time consuming. Since method D yielded 28 times the amount of DNA per preparation with comparable cost, it would be the method of choice for cells that are easy to transfect.

For cells more difficult to transfect, we used VERO and 3T3 cells to compare transfection efficiency of pHSVlac purified by each method. The results, shown in the table, demonstrate that transfections with pHSVlac prepared using method D result in higher b-galactosidase activity than using methods A, B or C. When the number of blue cells was counted, methods A, B and D were comparable. Because of the discrepancy between the yield of pHSVlac prepared using method C as measured by absorba
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