Preparation for Transformation
OverExpress™ Chemically Competent Cells are provided in aliquots of 40 μL sufficient for one transformation reaction.
Transformation is performed by heat shock at 42 °C, followed by incubation on ice.
To ensure successful transformation results, the following precautions must be taken:
- For best results, use a minimum of 1 μL of miniprep DNA (10-50 ng) for transforming OverExpress Chemically Competent Cells.
- The cells must be completely thawed on ice before use.
- For highest transformation efficiency, use the provided Expression Recovery Medium to resuspend the cells after transformation. Use of TB or other media may result in lower transformation efficiencies and induction of protein expression.
- Perform the heat shock in a chilled 15 mL disposable polypropylene culture tube (17 x 100 mm). The use of other types of tubes may dramatically reduce transformation efficiency.
- Prepare nutrient agar (LB-Lennox, YT) plates with antibiotic for selection. Remove Recovery Medium from the freezer and bring to room temperature.
- Remove OverExpress cells from the -80 °C freezer and thaw completely on wet ice (10-15minutes).
- Add 1 μL of DNA (10-50 ng) to the OverExpress 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 tube of OverExpress cells. Stir briefly.
- Important: Transfer the mixture of cells and DNA to a pre-chilled disposable polypropylene 15-mL culture tube (17 x 100 mm).
- Incubate culture tube(s) containing cells and DNA 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 small tube in which the cells are provided will significantly reduce the transformation efficiency.
- Return the tube of cells to ice for 2 minutes.
- Add 950 μL of room temperature Recovery Medium to the cells in the culture tube.
- Place the tube 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.
Sample Induction Protocol
- Inoculate a single colony from a freshly streaked plate into 5 mL of LB medium containing the appropriate antibiotic for the plasmid and host strain. For OverExpress pLysS strains, add chloramphenicol to 34 μg/mL, in addition to the antibiotic used for selection of the expression vector.
- Incubate with shaking at 37 °C overnight. To minimize the amount of expression of the target protein prior to induction, add glucose to the growth medium at a concentration of 0.2% (w/v).
- Inoculate 50 mL of LB medium containing the appropriate antibiotic with 0.5 mL of the overnight culture prepared in step 2.
- Incubate with shaking at 37 °C until the OD600 reaches 0.8-1.0.
- Add IPTG to a final concentration of 1 mM. Optimal time for induction of the target protein may vary from 2-16 hours, depending on the protein.
- Incubate at 37 °C for 3-4 hours. To determine the optimal time for induction of the target protein, it is recommended that a time course experiment be performed varying the induction from 2-16 hours.
- Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000 x g for 10 minutes at 4 °C.
- Remove the supernatant and store the cell pellet at -20 °C (storage at lower temperatures is also acceptable).