Price range: $145.00 through $475.00
Intact Genomics (IG®) C43(DE3) Chemically Competent Cells are optimized for the expression of toxic and difficult proteins from vectors with an IPTG-inducible T7 promoter. When protein expression gets tough, choose IG C43(DE3) competent cells.
Custom formats (8-well strip tube, 96 or 384 well plate), package sizes, and bulk pricing are available upon request.
Intact Genomics (IG®) C43(DE3) Chemically Competent Cells are optimized for transformation and routine recombinant protein expression from vectors driven by an IPTG-inducible T7 promoter.
C43(DE3) is a mutant derivative of BL21(DE3), further selected from C41(DE3) survivor cells that were able to overexpress the toxic subunit b of E. coli F-ATPase (Ecb). Compared to C41(DE3), C43(DE3) can express a distinct set of toxic proteins and carries additional mutations that help prevent cell death associated with the expression of many recombinant toxic proteins.
IG C43(DE3) is an E. coli BL21(DE3) mutant strain particularly effective for expressing toxic proteins from a wide range of organisms, including viruses, bacteria, yeasts, plants, animals, and mammals. The performance of the C43(DE3) strain in toxic protein expression has been validated by a patent and supported by many publications
Specifications:
Competent cell type: Chemically competent
Species: E. coli
Strain: C43(DE3)
Format: Tubes. Custom formats and package sizes available upon request.
Transformation efficiency: ≥ 3.0 x 107 cfu/µg pUC19 DNA
Blue/white screening: No
Shipping condition: Dry ice
Reagents Needed for One Reaction:
IG® C43(DE3) Chemically Competent Cells: 50 µl
DNA (or pUC19 Control, 10 pg/µl): 1 µl
Recovery medium: 1 ml
Product Components and Recommended Storage Condition:
Genomic Features:
IG® C43(DE3) chemically competent cells have the following features:
Quality Control:
Transformation efficiency is tested by using the pUC19 control DNA supplied with the kit and the high efficiency transformation protocol listed below. Transformation efficiency should be ≥ 3 x 107 CFU/µg pUC19 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.065 1065-
General Guidelines:
Follow these guidelines when using IG® C43(DE3) Chemically Competent Cells.
1047 1047-06 1047-24
| µl | 6×50µl, 24×50µl, 6×200µl, 12×200µl, 96×50µl (12 8-well strip), 1×96 well plate (20µl/well), 1×384 well plate (15µl/well) |
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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 = 2.0×1010
High-Efficiency Transformation Protocol:
Use this procedure to transform IG® C43(DE3) Chemically Competent Cells.
These cells are designed for chemical transformation only. Do NOT use for electroporation. For electroporation transformation, use IG® C43(DE3) Electro Competent Cells.
1. DNA/Cell Mixture Preparation
1.1. Remove competent cells from the –80 °C freezer and thaw completely on wet ice for 10–15 minutes. Ensure that Recovery Medium is readily available at room temperature.
1.2. Aliquot 1–5 µl of DNA (1 pg–100 ng) into each pre-chilled microcentrifuge tube on ice.
1.3. Once thawed, gently add 50 µl of competent cells to each DNA tube while keeping on ice. Mix gently by tapping the tube 4–5 times. If using the 50 µl package size competent cells, add DNA directly to the tube and mix gently by tapping.
Do NOT pipette up and down or vortex. Harsh mixing can damage cells and reduce transformation efficiency.
2. Chemical Transformation
2.1. Incubate the DNA/cell mixture on ice for 30 minutes.
2.2. Heat shock the cells by placing the culture tubes in a 42 °C water bath for 45 seconds.
2.3. Immediately return the tubes back to ice for 2 minutes.
3. Cell Recovery
3.1. Aliquot 950 µl of room-temperature Recovery Medium (or other suitable medium) into each culture tube (17 mm × 100 mm), then transfer the heat-shocked cell/DNA mixture into the corresponding tube.
3.2. Incubate the tubes in a shaking incubator at 37 °C by shaking at 210 rpm for 1 hour.
4. Cell Plating
4.1. Plate 50–200 µl of each transformation onto pre-warmed LB agar plates containing the appropriate antibiotic(s). Spread evenly using a sterile spreader or autoclaved ColiRoller™ plating beads.
Before cell plating, the plates should be prewarmed to growth temperature (37 °C), and be free of condensation to prevent contamination and mixed colonies.
We recommend plating two different volumes to ensure well-isolated colonies on at least one plate.
4.2. Incubate the plates overnight (12-16 hr) at 37 °C.
Five Minute Transformation Protocol:
The following procedure results in only ~10% of the transformation efficiency as the protocol listed above.
References:
