The different post-ligation treatments were also studied using cDNA ligated to a plasmid vector. Plasmids containing cDNA inserts (total size of 4.6 to 6.4 kb) were introduced into E. coli JS5 cells by electroporation, and transformation efficiencies were monitored as described previously. Again, heat treatment of the ligation reaction resulted in a 3- to 6-fold increase in electro-transformation efficiency, depending on the dilution (Figure 2). The heat-treated, 1/100 dilution samples generated the highest transformation efficiencies observed for the various diluted samples, but ethanol precipitation resulted in the highest efficiencies overall. The lower efficiencies seen with the cDNA samples when compared to the re-ligated pUC18 plasmid are most likely due to inaccuracy in determining the actual amount of cDNA that was ligated to the plasmid vector. Efficiencies were calculated assuming that all 10 ng of cDNA had ligated to the plasmid. Agarose gel electrophoresis revealed that 9 of 12 colonies contained cDNA inserts larger than 1,200 bp (data not shown).
The combination of post-ligation heat treatment at 65 C for 15 minutes followed by dilution to 1/100 with sterile water or ethanol precipitation (with glycogen) resulted in the highest E. coli transformation efficiencies with ligation mixtures. While ethanol precipitation of DNA from ligation mixtures generates high efficiencies, precipitating at