Competent Cells
For standard cloning experiments competent cells are available in two formats: high efficiency at greater than 108cfu/μg for cloning (JM109, HB101), and subcloning efficiency at greater than 107cfu/μg (HB101). JM109 cells have the additional feature of blue/white screening for recombinants.
For protein expression, JM109(DE3), BL21(DE3) or KRX competent cells can be used. The Single Step (KRX) Competent Cells are designed for efficient transformation and tightly controlled protein expression. These cells consolidate the best attributes of these two steps into one strain to evaluate protein expression in E. coli.
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What are Competent Cells?
Transformation of bacteria with plasmids is important because bacteria are used as the means for both storing and replicating plasmids. E. coli cells are more likely to incorporate foreign DNA if their cell walls are altered so that DNA can pass through more easily. Such cells are said to be "competent."
There are two primary methods for transforming bacterial cells: use of chemically competent cells and electroporation. Chemically competent cells are created using a series of cold salt washes to disrupt the cell membranes, preparing the cells to accept plasmid DNA. To prepare electrocompetent cells, the cells are chilled and washed with cold deionized water and glycerol.
To introduce the desired plasmid into chemically competent cells, the plasmid DNA is mixed with chilled cells and incubated on ice to allow the plasmid to come into close contact with the cells. The plasmid-cell mixture then is briefly heated to 45–50°C, allowing the DNA to enter the cell through the disrupted membrane. The heated mixture is then placed back on ice to retain the plasmids inside the bacteria.
For electroporation, the competent cells also sit on ice with the plasmid DNA. However, the plasmid-cell mixture is exposed to an electrical current, opening pores in the cell membrane so that the plasmid can enter the cell.
Both methods allow efficient recovery of transformed cells using antibiotic selection for the plasmid of interest.
