The optimization system for preparation of TG1 competent cells and electrotransformation

Certain parameters for the preparation of TG1 competent cells include optimum culture time of monoclonal bacteria, amplification growth concentration, and culture volume. The optimized transformation efficiency of TG1 ( ≥8 × 1010 cfu/μg DNA) was observed under suitable electric voltage (2.5 kV), electric intensity (15 kV/cm), and electric time (3.5 ms) of electricity for plasmid transformation. Optimized DNA amount (0.01–100 ng) dissolved in water led to the high efficiency of plasmid transformation but had low effi ciency when dissolved in the T4 ligation buffer. AbstractAn efficient electrotransformation system that includes electrocompetent cells is a critical component for the success of large ‐scale gene transduction and replication. The conditions of TG1 competent cell preparation and optimal electrotransformation were evaluated by investigating different parameters. Certain parameters for preparation of TG1 competent cells (≥8 × 1010 colony forming units (cfu)/ μg DNA) include optimum culture time of monoclonal bacteria (8–10 hr), amplification growth concentration (approximately OD600 = 0.45), and culture volume (400 ml in 2 L conical flask). With increased storage of competent cells at −80°C, electrotransformation efficiency gradually decreased, but it remains greater than ≥ 1010 cfu/μg DNA 3 months later. Moreover, the recovery time of electrotransformation also influenced electrotransformation efficiency (1.5–2 hr for optim...
Source: MicrobiologyOpen - Category: Microbiology Authors: Tags: ORIGINAL ARTICLE Source Type: research