![]() Cells should not be frozen or stored in liquid nitrogen, as this practice drastically reduces viability.įor consistency and to save time, premade competent cells are available in ready-to-use formats from commercial sources. Competent cells should remain stable for approximately 6–12 months when stored at –70☌ with minimal temperature fluctuations. The results are expressed as the number of colonies formed (transformants), or colony forming units (CFU), per microgram of plasmid DNA used (CFU/μg) (see cell plating).įor storage, aliquoting prepared cells in single-use volumes in screw-cap microcentrifuge tubes is recommended since each freeze/thaw cycle lowers transformation efficiency by about half. The transformation efficiency of competent cells is usually measured by the uptake of subsaturating amounts of a supercoiled intact plasmid (e.g., 10–500 pg of pUC DNA). Once prepared, competent cells should be evaluated for transformation efficiency, aliquoted to small volumes to minimize freeze/thaw cycles, and stored at an appropriate temperature to maintain viability. After 3 to 4 washes, the cells are finally pelleted and resuspended in 10% glycerol for storage. Electroporation: The harvested cells are washed with ice-cold deionized water several times by repeated pelleting and resuspension to remove salts and other components that may interfere with electroporation.To further improve competency, Ca 2+ may be supplemented or substituted with other cations and reagents, such as manganese (Mn 2+), potassium (K +), cobalt ( 3+), rubidium (Rb +), dimethyl sulfoxide (DMSO), and/or dithiothreitol (DTT), as described by Hannah et al. Heat-shock transformation: Competent cells are chemically prepared by incubating the cells in calcium chloride (CaCl 2) to make the cell membrane more permeable.Harvested cells are then processed according to the method of transformation, whether by heat shock or electroporation ( Figure 2). It is recommended that once the cells are harvested for further processing, all samples, reagents, and equipment be kept at 0–4☌ in order to improve cell viability and maintain transformation efficiency. In all steps, care must be taken to use sterile tools and labware, media, and reagents where appropriate or required. To obtain high transformation efficiency, it is crucial that cell growth be in the mid-log phase at the time of harvest-which generally occurs at OD 600 between 0.4 and 0.9, with the optimal value depending on the culture volume, strain, and protocol. This starter culture and the subsequent larger culture are carefully monitored for active growth by continually measuring optical density at 600 nm (OD 600). ![]() In either scenario, a single fresh colony of the desired strain is taken from an agar plate and inoculated into liquid medium for a starter culture ( Figure 2). The protocols for preparing competent cells vary by whether transformation is to be achieved via heat shock or electroporation. ![]() coli is very low or even nonexistent, the cells need to be made competent for transformation by heat shock or by electroporation. coli is the most common bacterial species used in the transformation step of a cloning workflow. ![]()
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