Plasmid DNA Transformation in Escherichia Coli: Effect of Heat Shock Temperature, Duration, and Cold Incubation of CaCl_2 Treated Cells

摘要

Various parameters of standard CaCl2/heat shock method on transformation of Escherichia coli strain DH5a -T1~R with plasmid pUC19 were optimized. Of the four different heat shock temperatures (32°C, 37°C, 42°C and 47°C) studied, 42°C treatment exhibited maximum efficiency of transformation as revealed by ampicillin-resistant colonies appearing on LB Agar ampicillin plates. Of the five different heat shock exposure times, a pulse of 30 second duration combined with 42°C heat shock temperature exhibited maximum efficiency. It was observed that although transformation of CaCl_2 treated cells occurs even before heat shock treatment, the efficiency was -15 fold higher after heat shock. When the cells were further incubated on ice (after heat shock)for 10 min, the transformation efficiency increased by 24 fold compared to no heat shock and 1.6 fold compared to heat shock treatment. There was a marginal decrease in transformation when cells were incubated at room temperature instead of ice after heat shock. These results suggest that a heat shock pulse of 30 sec at 42°C followed by a 10 min ice incubation step are ideal parameters to obtain maximum transformation efficiency in DH5a -Tl strain. Results also suggest that post heat shock cold incubation step is also an important factor and enhances transformation of E. coli significantly.