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High Electrotransformation Efficiencies Obtained With DNA From Ligation Mixtures

C medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was added to the cuvettes with a thintipped disposable pipette. The cells were gently resuspended, transferred to a test tube, and incubated without shaking at 37 C for 1 hour. The cells were diluted 1/10, 25 - 50 l of the dilution were plated onto LB-ampicillin plates (100 g/ml), and the plates were inverted and incubated at 37 C overnight.

Results and Discussion
It has been shown that several components of ligation mixtures have an inhibitory effect on transformation.5 Therefore there is a need to either remove or reduce the concentration of ligation components by alcohol precipitation or dilution before electroporation in order to achieve optimal transformation efficiencies. It also has been previously shown that heat treatment of the ligation reactions increases the electro-transformation efficiency.5 I investigated this by re-ligating a linear pUC18 plasmid and subjecting this ligation reaction to combinations of heat treatment, 1/10 and 1/100 dilutions, and alcohol precipitations, followed by electro-transformation. I observed that heat treatment of the ligation reaction resulted in a 2- to 5-fold increase in transformation efficiency over the non-heat treated ligation mixture, depending on the dilution factor (Figure 1). Both dilution to 1/100 and ethanol precipitation of the heat-treated ligation mixture in the presence of glycogen achieved nearly the same high electro-transformation efficiency as did the control supercoiled pUC18 plasmid (Figure 1). Glycogen was used as a carrie
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