Ar.A4 Electrocompetent Agrobacterium cells are made from a specific strain of Rhizobium rhizogenes (formerly Agrobacterium rhizogenes), Agrobacterium rhizogenes(kan R) Ar A4 Ri(agropine type). Agrobacterium rhizogenes is a soil-borne gram-negative bacterium that can infect most dicotyledons, a few monocotyledons, and some gymnosperms. Ar.A4 Electrocompetent Agrobacterium are optimized for the highest transformation efficiencies and are useful for transgenic operations of corn, tobacco, carrot, and other plants. Ar.A4 Agrobacterium rhizogenes strain contains agrobacterium-type Ri plasmid and displays kanamycin resistance.
Specifications:
Competent cell type: Electrocompetent
Species: R. rhizogenes
Strain: Ar.A4
Format: Tubes
Transformation efficiency: ≥ 1 x 107 cfu/µg pIG7spe DNA
Blue/white screening: No
Shipping condition: Dry ice
Reagents Needed for One Reaction:
Ar.A4 ElectroCompetent Agrobacterium: 25 µl
DNA (pIG7-spe, 500 pg/µl): 1 µl
Recovery medium: 1 ml
Storage:
Ar.A4 ElectroCompetent Agrobacterium: -80ºC
pIG7-spe control DNA: -20ºC
Recovery medium: 4ºC
Quality Control:
Transformation efficiency is tested by using the pIG7-spe control DNA supplied with the kit and using the protocol in this manual. Transformation efficiency should be ≥1 x 107 CFU/µg pIG7-spe DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.
General Guidelines:
Follow these guidelines when using Ar.A4 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.
Calculation of Transformation Efficiency:
Transformation 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/Plated
Transform 1 µl of (500 pg/µl) pIG7-spe 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 = 500
µg of DNA = 0.0005
Dilution = 100/1000 = 0.1
TE = 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.