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Team:University of Ottawa/Transformation of Competent Ecoli
From 2008.igem.org
Transformation of Competent Cells
This protocol allows for fast and relatively efficient transformation of competent E. coli cells stored in TSS. All steps performed at the workbench should be accompanied by a flame for sterility. An alternate protocol that includes a heat shock treatment is also provided, but has not been shown to improve efficiency.
- Thaw competent cells (DH5α, XL10, etc) stored at –80$deg;C on ice.
- Set water bath or heat block to 42°C, remove appropriate number of agar plates with correct antibiotic selection from the cold room to adjust to room temperature. If plate is wet, incubate at 37°C incubator.
- Add a maximum of 20 μl of ligation mix (~50 ng of vector with insert) to a labeled tube containing the aliquoted competent cells (maintain cells on ice). An effort should be made to minimize the volume of DNA.
- Add 100 μL (lab grown) or 50 μL (commercial) of competent cells to each tube. Mix by gently pipetting up and down with a wide bore pipet tip.
- Set tubes on ice for 20 minutes.
- Place tubes in the 42°C water bath or heat block for 45 to 90s to allow entry of DNA by “heat shock”.
- Set tubes back on ice for at least 5 min.
- Add 900 μl of LB broth to each tube of competent cells.
- Incubate competent cells in shaker for 45 min at 37°C and 200 RPM.
- Spin tubes for about 2 minute at 6,000 RPM (max setting) to pellet cells.
- Discard all but 100μl of supernatant.
- Resuspend cells in remaining supernatant by gently pipetting the mixture up and down, while trying not to create bubbles.
- Pipette cell suspension onto LB agar plate with appropriate antibiotic selection conditions. Immerse spreader in 100% ethanol, pass it through flame and allow ethanol to burn off. Allow spreader to cool by making contact with the LB/agar away from the cell suspension with a back and forth movement for 1-2s. Spread cells evenly around with sterile spreader.
- Allow cells to absorb into gel at room temperature on bench top for at least 30 min.
- Incubate plates upside down at 37°C overnight to prevent condensation from settling on the agar gel.
