Intact Genomics 10B Copy-up BAC ElectroCompetent E. coli cells offer the highest transformation efficiencies of ≥5 x 1010 cfu/µg plasmid DNA which are ideal for applications requiring high transformation efficiencies, such as with cDNA or gDNA library construction. The cells contain a mutant trfA gene, whose protein product is required for initiation of replication from the oriV origin of replication. The trfA gene is under control of an inducible promoter. When grown in standard LB or SOC medium, expression of the trfA gene is repressed. Addition of L-arabinose induces expression of the trfA gene and subsequent utilization of the oriV origin of replication and high copy amplification of the Copy-Up BAC, fosmid and PCR clones.
Competent cell type: ElectroCompetent
Derivative of: DH10B™
Species: E. coli
Transformation efficiency: ≥ 5 x 1010 cfu/µg pUC19 DNA
Inducible Promotor: Yes
Blue/white screening: Yes
Shipping condition: Dry ice
Reagents Needed for One Reaction
10B Copy-up BAC ElectroCompetent Cells: 20 µl
DNA (or pUC19 Control, 10 pg/µl): 1 µl
Recovery medium: 1 ml
10B Copy-up BAC ElectroCompetent Cells: -80 ºC
pUC19 control DNA: -20 ºC
Recovery medium: 4 ºC
10B Copy-up BAC ElectroCompetent Cells have the following features:
- Mutant trfA gene under control of an inducible promoter for complete copy number control of Copy-up clones.
- Φ80lacZΔM15 marker provides α-complementation of the β-galactosidase gene with blue/white screening.
- mcrA genotypic marker and the mcrBC, mrr deletion allows for cloning DNA that contains methylcytosine and methyladenine.
F – mcrA ∆(mrr-hsdRMS-mcrBC) endA1 recA1 φ80dlacZ∆M15 ∆lacX74 araD139 ∆(ara, leu)7697 galU galK rpsL (StrR) nupG λ-
Transformation efficiency is tested by using the pUC19 control DNA supplied with the kit and using the protocol given below. Transformation efficiency should be ≥5 x 1010 CFU/µg pUC19 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.
Follow these guidelines when using BAC 10B 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 x 1010