Competent E. coli cells

Competent E. coli cells (high quality is required > 109 transformants/pg su-percoiled DNA). 

Incubate the cells with these samples on ice for 15 min, swirling gently every 2 min. Quickly remove the tubes from the ice and place them in a 42 °C water bath. Incubate the tubes at 42 °C for exactly 45 sec. Quickly remove the tubes from the water bath and incubate them again on ice for 2 min. On heat shock, the competent E. coli cells will take up the intact plasmid DNA produced during the ligation reaction. The competent cells are very compromised at this point, and they will die if the incubation is not performed at exactly 42°C for exactly 45 sec. The incubation on ice will prevent the cells from dying after the heat shock. 

Hartley et al. (1986) devised a nonradioactive probe, called probe-vector, which can, after hybridization, transform competent E. coli cells and, depending on the transformation efficiency, detect as little as 0.1 pg of target nucleic acid. The probe-vector molecules are linear, partially si DNA prepared by hybridizing individually prepared DNA strands. The ds region of the probe-vector encodes a phenotypic marker and origin of replication. The two terminal si... 

We routinely use TOPIO chemically competent E. coli cells (Invitrogen, Carlsbad, CA) however, other strains, such as DH5a, should work. 

Transform pMarGent or pGKT (see Fig. 1) into competent E. coli cells and plate in the presence of the appropriate antibiotic (i.e., gentamicin for pMarGent or gentamicin and kanamycin for pGKT). 

Remove about 8 pL of digested DNA and ligate in 10 pL volume at 14 °C for 6 h (or overnight). Transform entire ligation into chemically competent E. coli cells and plate about half of the transformed cells on LB plates containing gentamicin. Incubate plates at 37 °C overnight. 

For preparation of competent E. coli cells, we routinely use the procedure described previously (15). In this procedure, E. coli cells are grown at 18°C prior to harvesting and preparation of the competent cells. 

Expression of the 8-3 Mutant of GpdQ from competent E. Coli cells was conducted at the University of Queensland using same protocol as for wt-GpdQ. The plasmid was obtained from Prof. David Ollis, Research School of Chemistry, ANU, Canberra. 

Z-Competent E. coli cells (Zymo Research, Orange, CA), store at <70°C. 

Use the ligation mixture to transform into competent E. coli cells (see Subheading 3.7). 

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