BL21 Chemically Competent E. coli Cells

GoldBio’s BL21 Chemically Competent E. coli cells are a good, universal strain of cell that is gives great transformation for protein expression and induction, and is especially useful for non-T7 vectorsprotein expression systems. BL21 chemically competent cells feature a widely used host background, and are deficient in both lon (1) and ompT proteases. In addition, BL21 Chemically Competent E. coli cells are resistant to phage T1 (fhuA2).

BL21 chemically chemically competent cells are free of animal-derived products and grown with animal-free media.

BL21 Highlights

• Free of all Animal products
• High plasmid stability and decreased plasmid transfer
• No DNA cytosine methylation means better restriction enzyme digestion
• No restriction/methylation system
• Galactose deficiency and ideal for selective systems
• Perfect for maltose-based T7-based systems
• Sensitive to phage λ (No T7 polymerase present)
• Ideal for stable protein expression with low protease activity.

Kit Components

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

Reagents Needed for One Reaction

• BL21 chemically competent cells: 50 µL
• DNA (or pUC19 Control, 500 pg/µL): 2 µL
• Recovery medium: 1 mL

Storage/Handling

This product may be shipped on dry ice. BL21 Chemically Competent 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 10⁶ cfu/µg efficiency
• Widely used host background
• Routine non-T7 vector expression.
• Deficient in both lon (1) and ompT proteases.
• Resistant to phage T1 (fhuA2).

Genotype

F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)

Applications

• High-level recombinant protein expression
• Expression of proteins with minimal degradation
• Production of tagged proteins (His-tag, GST-tag) for purification
• Use in T7 promoter-based systems
• Production of inclusion bodies for insoluble proteins
• Isotope-labeled protein production for NMR or mass spectrometry
• Transformation of unmethylated or methylated plasmids

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 10⁶ 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.

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