BL21 Chemically Competent Cells Overview

Preparation for Transformation

BL21 Chemically Competent Cells are transformed in 40 μL reactions. Cells are packaged with sufficient volumes for 1, 2, or 4 reactions per tube.

Transformation is performed by heat shock at 42 °C, followed by incubation on ice.

To ensure successful transformation results, the following precautions should be taken:

• All 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 Expression 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 the transformation efficiency.

Transformation Protocol

1. Prepare nutrient agar (LB-Lennox or YT) plates with antibiotic for selection. Remove Recovery Medium from the freezer and bring to room temperature.
2. Chill sterile culture tubes on ice (17 mm x 100 mm tubes, one tube for each transformation reaction).
3. Remove cells from the -80 °C freezer and thaw completely on wet ice (5-15 minutes).
4. Add 40 μL of cells to the chilled culture tube.
5. Add 1 μL of DNA 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 culture tube containing 40 μL of cells. Stir briefly.
6. Incubate the cell/DNA mixture on ice for 30 minutes.
7. 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.
   
   
8. Return the tubes to ice for 2 minutes.
9. Add 960 μL of room temperature Expression Recovery Medium to the cells in the culture tube.
10. Place the tubes in a shaking incubator at 250 rpm for 1 hour at 37 °C.
11. 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 200 μL of the transformation on LB-Lennox or YT agar plates containing 100 μg/mL carbenicillin or ampicillin.
12. Incubate the plates overnight at 37 °C.
13. Transformed clones can be further grown in LB or any other lactose-minus medium.
    

Sample Induction Protocol

1. 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.
2. Incubate with shaking at 37 °C overnight. To minimize the expression of the target protein prior to induction, add glucose to the growth medium to a final concentration of 0.2% (w/v).
3. Inoculate 50 mL of LB medium containing the appropriate antibiotic with 0.5 mL of the overnight culture prepared in step 2.
4. Incubate with shaking at 37 °C until the OD600 reaches 0.6 - 0.8.
5. Add IPTG to a final concentration of 1 mM. To determine the optimal concentration of IPTG for maximum expression of the target protein test a range of IPTG concentrations from 0.25 – 2 mM.
6. Incubate at 37 °C for 3-4 hours. The optimal time for induction of the target protein may vary from 2-16 hours.
7. Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000 x g for 10 minutes at 4 °C.
8. Remove the supernatant and store the cell pellet at -20 °C (storage at lower temperatures is also acceptable).