BL21 (DE3) Chemically Competent Cells

Description

Intact Genomics chemically competent BL21(DE3) E. coli cells are suitable for transformation and routine protein expression.

Specifications

Competent cell type:                Chemically competent
Derivative of:                            BL21(DE3)
Species:                                   E. coli
Format:                                    Tubes
Transformation efficiency:         ≥1.0 x 10⁹ cfu/µg pUC19 DNA
Blue/white screening:               Yes
Shipping condition:                   Dry ice

Reagents Needed for One Reaction

BL21(DE3) Chemically Competent Cells:             50 µl
DNA (or pUC19 Control, 10 pg/µl):                         1 µl
Recovery Medium:                                                 1 ml

Storage

BL21(DE3) Competent Cells:                -80 ºC
pUC19 Control DNA:                            -20 ºC
Recovery Medium:                                  4 ºC

Genomic Features

BL21(DE3) chemically competent cells have the following features:

• 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 high efficiency transformation protocol listed below. Transformation efficiency should be ≥ 1×10⁹ CFU/µg pUC19 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.

General Guidelines

Follow these guidelines when using BL21(DE3) chemically competent E. coli.

• 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 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 = 2.0×10¹⁰1051-06 1051-12 1052-12 1052-24 1054-24 1054-48