Useful for Agrobacterium-mediated genetic transformation of corn, tobacco, carrot and other plants.
Ar.A4 Chemically Competent Agrobacterium
$355.00 – $755.00
Intact Genomics (ig®) Ar.A4 Chemically Competent 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 Chemically Competent 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.
Competent cell type: Chemically Competent
Species: R. rhizogenes
Transformation efficiency: ≥ 1 x 105 cfu/µg pIG7spe DNA
Blue/white screening: No
Shipping condition: Dry ice
- Ar.A4 Chemically Competent Agrobacterium
- DNA (pIG7spe, 500 pg/µl)
- Recovery medium
Note: Liquid nitrogen is required.
Ar.A4 Chemically Competent Agrobacterium: -80 ºC
pIG7spe control DNA: -20 ºC
Recovery medium: 4 ºC
Transformation efficiency is tested by using the pIG7spe control DNA supplied with the kit and using the protocol in this manual. Transformation efficiency should be ≥1 x 105 CFU/µg pIG7spe DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.
Follow these guidelines when using Ar.A4 Chemically Competent Agrobacterium 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.
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) pIG7spe control plasmid into 50 µl of cells, add 950 µl of Recovery Medium. Recover for 3 hours and plate 100 µl. Count the colonies on the plate in two days. If you count 5 colonies, the TE is calculated as follows:
Colonies = 5
µg of DNA = 0.0005
Dilution = 100/1000 = 0.1
TE = 5/.0005/.1 = 1×105
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.1072-06 1072-18
Use this procedure to transform Ar.A4 Chemically Competent Agrobacterium cells . Do not use these cells for electro competent transformation.
1) Place 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 50μl of cells to each DNA tube on ice and mix gently by tapping 4-5 times. For the pIG7spe control, add 1 µl of (500 pg/µl) DNA to the 50 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) Keep tubes on ice for 5 minutes, and then transfer to liquid nitrogen for 5 minutes.
6) Incubate tubes for additional 5 minutes in 37°C water bath.
7) Immediately add 950µl of Recovery Medium or any other medium of choice to the tube, pipette up and down three times to re-suspend the cells.
8) Incubate tubes at 30 °C for 3 hours at 200 RPM.
9) Dilute the cells as appropriate then spread 20-200 μl cells onto a pre-warmed selective plate. For the pIG7spe 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.
10) Incubate the plates for 2 – 3 days at 30 °C.