- Description Intact Genomics 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. Specifications Competent cell type: Electrocompetent Derivative of: BL21 (DE3) Species: E. coli Format: Tubes Transformation efficiency: ≥1 x 1010 cfu/µg pUC19 DNA Blue/white screening: No Shipping condition: Dry ice Reagents Needed for One Reaction BL21(DE3) Electrocompetent Cells: 25 µl DNA (or pUC19 Control, 10 pg/µl): 1 µl Recovery medium: 1 ml Storage BL21(DE3) Electrocompetent Cells: -80 ºC pUC19 control DNA: -20 ºC Recovery medium: 4 ºC Genomic Features BL21(DE3) Electrocompetent Cells have the following features: >1 x 1010 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) 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 1010 CFU/µg pUC19 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity. General Guidelines Follow these guidelines when using BL21(DE3) Cells: 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 such as Bio-Rad Gene Pulser II #165-2105, 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 1µg of plasmid into a given volume of competent cells. TE = Colonies/µg/Dilution Transform 1 µl of (10 pg/µl) pUC19 control plasmid into 50 µl of cells, add 950 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 100 colonies, the TE is calculated as follows: Colonies = 100 µg of DNA = 0.00001 Dilution = 50/1000 x 10/1000 = 0.0005 TE = 100/.00001/.0005 = 2.0×10101252-12 1252-24 1254-24 1254-48
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