Intact Genomics (ig®) Electrocompetent BL21(DE3)pLysS electrocompetent cells are suitable for transformation and routine protein expression. The pLysS plasmid produces T7 lysozyme that reduces base level expression of the gene of interest, subsequently allowing for tighter control of expression and is therefore suitable for expression of toxic genes. BL21(DE3)pLysS electrocompetent cells also carry the chloramphenicol resistance gene.
Competent cell type: ElectrocompetentDerivative of: BL21(DE3)pLysSSpecies: E. coliFormat: TubesTransformation efficiency: ≥1 x 1010 cfu/µg pUC19 DNABlue/white screening: YesShipping condition: Dry ice
Reagents Needed for One Reaction
ig® BL21(DE3)pLysS electrocompetent cells: 25 µlDNA (or pUC19 Control, 10 pg/µl): 1 µlRecovery medium: 1 ml
Storage
ig® BL21(DE3)pLysS electroCompetent cells: -80 ºCpUC19 control DNA: -20 ºCRecovery medium: 4 ºC
Genomic Features
ig® BL21(DE3)pLysS electrocompetent cells have the following features:
- T7 Expression Strain
- Deficient in both lon (1) and ompT proteases
- Resistant to phage T1 (fhuA2)
- B Strain
- Suitable for expression of toxic genes
Genotype
F–pLysS, Cmr 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 ig® BL21(DE3)pLysS ElectroCompetent 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.
Example Calculation of Transformation EfficiencyTransformation 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.1256-48
