Intact Genomics (ig^®) ccdB Resist™ Electrocompetent E. coli cells are suitable for high efficiency transformation in a wide variety of applications such as cloning and sub-cloning. ccdB Resist™ cells are capable of propagation of plasmids containing the ccdB gene. Intact Genomics ccdB Resist™ electrocompetent cells provide superb transformation efficiency, higher than any competitors similar product, allowing for increased opportunity for experimental success.

Custom Aliquots Available

Specifications

Competent cell type:  ElectroCompetentSpecies:  E. coliFormat: TubesTransformation efficiency:  ≥ 1 x 10¹⁰ cfu/µg pUC19 DNABlue/white screening:  NoShipping condition:  Dry ice

Reagents Needed for One Reaction

ig^® ccdB Resist™ electrocompetent cells:  25 µlDNA (or pUC19 Control, 10 pg/µl):  1 µlRecovery medium:  1 ml

Storage

ig^® ccdB Resist™ electroCompetent cells:  -80 ºCpUC19 control DNA:  -20 ºCRecovery medium:  4 ºC

Genomic Featuresig^®  ccdB Resist™ electrocompetent cells have the following features:

• ccdB Resist™ allows for cloning of methylated genomic sequences
• Stabilizes retroviral and direct repeat sequences including HIV
• High transformation efficiency allows aids in cloning rare sequences
• May be used for plasmids > 20 kb
• endA1 mutation increases plasmid yield significantly

Genotype

F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG fhuA::IS2

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^®  ccdB Resist™ 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.

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 = 100µg of DNA = 0.00001Dilution = 50/1000 x 10/1000 = 0.0005TE = 100/.00001/.0005 = 2.0×10¹⁰1269-12 1269-24