Intact Genomics (ig®) K599 Electrocompetent Agrobacterium cells are made from a specific strain of Agrobacterium rhizogenes, Agrobacterium rhizogenes (str R) pRi2659 (agropine type). K599 was originally isolated from cucumber exhibiting hairy root disease symptoms and has been widely used for hairy root transformation(1).
Agrobacterium rhizogenes is a soil-borne gram-negative bacterium that can infect most dicotyledons, a few monocotyledons, and some gymnosperms. K599 Electrocompetent Agrobacterium is optimized for the highest transformation efficiencies and is useful for transgenic operations of corn, soybean, cotton, peanut, dandelion, cowpea, and other plants. K599 Agrobacterium rhizogenes strain contains pRi2659 agrobacterium-type Ri plasmid and displays streptomycin resistance.
SpecificationsCompetent cell type: ElectrocompetentSpecies: A. rhizogenesStrain: K599Format: TubesTransformation efficiency: ≥ 1 x 105 cfu/µg pCAMBIA1391zBlue/white screening: NoShipping condition: Dry ice
Reagents Needed for One ReactionK599 ElectroCompetent Agrobacterium: 25 µlDNA (pCAMBIA1391z, 500 pg/µl): 1 µlRecovery medium: 1 ml
StorageK599 ElectroCompetent Agrobacterium: -80 ºCpCAMBIA1391z control DNA: -20 ºCRecovery medium: 4 ºC
Quality ControlTransformation efficiency is tested by using the pCAMBIA1391z control DNA supplied with the kit and using the protocol in this manual. Transformation efficiency should be ≥1 x 105 CFU/µg pCAMBIA1391z DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.
General GuidelinesFollow these guidelines when using K599 ElectroCompetent Agrobacterium:
- 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 the plasmid into a given volume of competent cells.TE = Colonies/µg/PlatedTransform 1 µl of (500 pg/µl) pCAMBIA1391z control plasmid into 25 µl of cells, and add 974 µl of Recovery Medium. Recover for 3 hours and plate 100 µl. Count the colonies on the plate in two days. If you count 500 colonies, the TE is calculated as follows:Colonies = 500µg of DNA = 0.0005Dilution = 100/1000 = 0.1TE = 500/.0005/.1 = 1×107
Please note, all agrobacterial strains are not well-studied for antibiotic resistance and there are many agrobacterial strains. Therefore, it is the customer’s responsibility to make sure his/her vectors are compatible with the Agrobacterial strains if he/she uses an alternate antibiotic selection than spectinomycin-selection.
References
- Combard, A.; Brevet, J.; Borowski, D.; Cam, K.; Tempé, J. Physical map of the T-DNA region of Agrobacterium rhizogenes strain NCPPB2659. Plasmid 1987, 18, 70–75.1272-
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