Preparation for Transformation
To ensure successful transformation results, the following precautions must be taken:
- For best results, ligation reactions must be heat inactivated at 70 ºC for 15 minutes before transformation. Alternately, the reactions may be purified.
- Prepare nutrient agar plus antibiotic for selection. We suggest using LB-Lennox or YT agar to achieve optimal cell growth.
- All microcentrifuge tubes must be thoroughly pre-chilled on ice before use.
- The cells must be completely thawed on ice before use.
- For highest transformation efficiency, use the provided Recovery Medium to resuspend the cells after transformation.
- Perform the heat shock in a 15 mL disposable polypropylene culture tube (17 x 100 mm). The use of other types of tubes may dramatically reduce transformation efficiency.
Transformation Protocol for SIG10 Cells
- Prepare nutrient agar plates (LB-Lennox or YT) with antibiotic for selection. Ensure that Recovery Medium is readily available at room temperature.
- Chill sterile culture tubes on ice (17 mm x 100 mm tubes, one tube for each transformation reaction).
- Remove to SIG10 cells from the -80 °C freezer and thaw completely on wet ice (5-15 minutes).
- Add 40 µL of cells to the chilled culture tube.
- Add 1-4 µL of DNA sample to the 40 µL of cells. Stir briefly with a pipet tip; do not pipet up and down to mix, which can introduce air bubbles and warm the cells. For the pUC19 control, add 1 μL (10 pg) of DNA to another chilled culture tube containing 40 μL of cells.
- Incubate the cell/DNA mixture on ice for 30 minutes.
- Heat shock cells by placing the culture tubes in a 42 °C water bath for 45 seconds.
Performing the heat shock in the 1.7 mL tube in which the cells are provided will significantly reduce the transformation efficiency.
- Return the culture tubes to ice for 2 minutes.
- Add 960 µL of room temperature Recovery Medium to the cells in the culture tube. When using these cells with a cloning kit, follow the Recovery Medium volume given in that kit manual.
- Place the tubes in a shaking incubator at 250 rpm for 1 hour at 37 °C.
- Plate up to 200 µL of the transformation on LB-Lennox or YT agar plates containing the appropriate antibiotic. The plating volume may need to be optimized depending on your DNA. For the pUC19 control, plate 25 μL of 10G cells or 50 μL 10GF’ cells on LB-Lennox agar plates containing 100 μg/mL carbenicillin or ampicillin. Transformants plated on LB-Miller may grow slowly.
- Incubate the plates overnight at 37 °C.
- Transformed clones can be further grown in any rich culture medium (e.g. LB or TB).