BL21 (DE3) Electrocompetent Cells

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:               Yes
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) 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.

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