The K599 strain is useful for transgenic operations of useful for transgenic operations of corn, tobacco, carrot and other plants.
K599 ElectroCompetent Agrobacterium
$350.00 – $750.00
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 are optimized for the highest transformation efficiencies and are 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.
Competent cell type: Electrocompetent
Species: A. rhizogenes
Transformation efficiency: ≥ 1 x 105 cfu/µg pCAMBIA1391z
Blue/white screening: No
Shipping condition: Dry ice
Reagents Needed for One Reaction
K599 ElectroCompetent Agrobacterium: 25 µl
DNA (pCAMBIA1391z, 500 pg/µl): 1 µl
Recovery medium: 1 ml
K599 ElectroCompetent Agrobacterium: -80 ºC
pCAMBIA1391z control DNA: -20 ºC
Recovery medium: 4 ºC
Transformation 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.
Follow 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 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/Plated
Transform 1 µl of (500 pg/µl) pCAMBIA1391z control plasmid into 25 µl of cells, 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.
- 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-
Use this procedure to transform K599 ElectroComp Agrobacterium. Do not use these cells for chemically transformation.
1) Place sterile cuvettes and microcentrifuge tubes on ice.
2) Remove competent cells from the -80 °C freezer and thaw completely on wet ice (10-15 minutes).
3) Aliquot 1 µl ( 10pg -1 µg) of DNA to the chilled microcentrifuge tubes on ice.
4) When the cells are thawed, add 25 μl of cells to each DNA tube on ice and mix gently by tapping 4-5 times. For the pIG7-spe control, add 1 µl of (500 pg/µl) DNA to the 25 µl of cells on ice. Mix well by tapping. Do not pipette up and down or vortex to mix, this can harm cells and decrease transformation efficiency.
5) Pipette 26 µl of the cell/DNA mixture into a chilled electroporation cuvette without introducing bubbles. Quickly flick the cuvette downward with your wrist to deposit the cells across the bottom of the well and then electroporate.
6) Immediately add 974 µl of Recovery Medium or any other medium of choice to the cuvette, pipette up and down three times to re-suspend the cells. Transfer the cells and Recovery Medium to an Eppendorf tube.
7) Incubate tubes at 30 °C for 3 hours at 200 RPM.
8) Dilute the cells as appropriate then spread 20-200 μl cells onto a pre-warmed selective plate. For the pCAMBIA1391z control, you may plate 100 μl of undiluted transformation mix onto a YT plate containing 100 μg/ml spectinomycin. Use a sterilized spreader or autoclaved ColiRoller™ plating beads to spread evenly.
9) Incubate the plates for 2 – 3 days at 30 °C.
Mode Exponential protocol
Voltage (V) 1,800 V
Capacitance 25 uFD
Resistance 200 Ohms
Cuvette 1 mm