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This protocol is based on the transformation protocol laid out in Sambrook and Russell. Transformed strains are only stable on plates for 7-10 days, therefore glycerol stocks or fresh transformations are required.
- ( Note: Due to the presence of recA and other DNA repair machinery in JS006 and derivatives, plasmids in these strains are less stable than in cloning strains such as DH10B. Therefore, glycerol stocks of networks in JS006 or derivative strains are less stable, and not a recommended means of maintaining these strains. Fresh transformations are a better option.)
- Remove a 100µl aliquot of competent cells from the -80°C freezer
- Let the aliquot thaw on ice for 5-10 minutes, or until all ice crystals have melted
- Add 100µl of cells to a pre-chilled 14ml polypropelene tube on ice.
- Add 1µl (~50-100ng) pf plasmid DNA to the cells and mix gently.
- Incubate on ice for 30 min.
- Place at 42°C for 45 seconds.
- Incubate on ice for 2 min.
- Add 250µl of SOC media to the tube (See Protocols for recipe).
- Incubate at 37°C with shaking for 1 hour
- Note: This step can be shortened or even omitted entirely if the plasmid used contains an ampicillin resistance cassette, but is required for other selective markers
- Plate 250µl of cell mixture on LB/agar plates with appropriate antibiotic(s).
- Incubate at 37°C overnight.
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