BL21 (DE3) Electrocompetent E. coli Cells

GoldBio’s BL21 (DE3) Electrocompetent E. coli cells are suitable for high efficiency transformation and routine protein expression. Increased cloning efficiencies versus typical BL21 cells makes the BL21 (DE3) Electrocompetent E. coli cells ideal for construction of complex expression libraries and feature ≥1 x 10⁸ cfu/µg efficiency with electroporation. BL21 (DE3) Electrocompetent E. coli cells have a widely used background, feature a T7 expression strain, are deficient in both lon (1) and ompT proteases. These cells are resistant to phage T1(fhuA2) and are B strain.

Kit Components

• Competent Cells
• 1 x 12 mL Recovery Media
• 1 x 10 µl Control Plasmid (pUC19 Control, 500 pg/µl)

Product Specifications
Competent cell type: ElectroCompetent
Derivative of: BL21 (DE3)
Species: E. coli
Format: Tubes
Transformation efficiency: ≥1 x 10⁸ cfu/µg pUC19 DNA
Blue/white screening: Yes

Storage/Handling: This product may be shipped on dry ice. BL21 (DE3) Electrocompetent E. coli cells should be stored at -80°C, pUC19 Control DNA should be stored at -20°C and recovery medium should be stored at 4°C immediately upon arrival. When stored under the recommended conditions and handled correctly, these products should be stable for at least 1 year from the date of receipt.

Genomic Features

• ≥1 x 10⁸ cfu/µg efficiency with electroporation
• Widely used host background
• T7 Expression Strain
• Deficient in both lon (1) and ompT proteases
• Resistant to phage T1 (fhuA2)
• B Strain

Genotype
F–ompT hsdS(rB– mB–) gal dcm λ(DE3)

Reagents Needed for One Reaction

• BL21 (DE3) electrocompetent cells: 25 µl
• DNA (or pUC19 Control, 10 pg/µl): 1 µl
• Recovery medium: 1 ml

Quality Control
Transformation efficiency is tested by using the pUC19 control DNA supplied with the kit and using the protocol given below. Transformation efficiency should be ≥1 x 10⁸ CFU/µg pUC19 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.

General Guidelines

• Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting.
• Thaw competent cells on ice, and transform cells immediately following thawing. After adding DNA, mix by tapping the tube gently. Do not mix cells by pipetting or vortexing.

Note: A high-voltage electroporation apparatus capable of generating field strengths of 16 kV/cm is required.

Calculation of Transformation Efficiency
Transformation Efficiency (TE) is defined as the number of colony forming units (cfu) produced by transforming 1 µg of plasmid into a given volume of competent cells.

• TE = Colonies/µg/Dilution
  • Colonies = the number of colonies counted
  • µg = amount of DNA transformed in µg
  • Dilution = total dilution of the DNA before plating

Example: Transform 1 µl of (10 pg/µl) control plasmid into 25 µl of cells, add 975 µl of Recovery Medium. Dilute 10 µl of this in 990 µl of Recovery Medium and plate 50 µl. Count the colonies on the plate the next day. If you count 250 colonies, the TE is calculated as follows:

Colonies = 250
µg of DNA = 0.00001
Dilution = 10/1000 x 50/1000 = 0.0005
TE = 250/0.00001/0.0005 = 5.0 × 10¹⁰