ig® Thymidine-Auxotrophic EHA101 Agro cells includes modifications so that they will not grow unless thymidine is added to Minimal medium. This prevents the bacteria from overgrowing plant tissues.
Thymidine Auxotrophic EHA101Thy Agrobacterium Electrocompetent Cells
$505.00 – $1,100.00
Intact Genomics (ig®) Thymidine-Auxotrophic EHA101Thy Agrobacterium ElectroCompetent Cells include modifications so that they will not grow unless 50 mg/L of thymidine is added to Minimal medium. This prevents the bacteria from overgrowing plant tissues when used for plant transformation. Thymidine-Auxotrophic EHA101Thy Agrobacterium ElectroCompetent Cells are optimized for the highest transformation efficiencies. The EHA101Thy strain is useful for transgenic operations of rice, tobacco and other plants. EAH101Thy– thyA knockout mutant, spectinomycin-resistant, derived from EHA101, itself a derivative of A281 (A136/pTiBo542).
Custom Aliquots Available
- Thymidine Auxotrophic
- Enables development of more efficient transformation systems
- Reduced bacterial overgrowth during co-cultivation
- Decreased need for antibiotics
Competent cell type: Electroporation Competent
Species: A. tumefaciens
Transformation efficiency: ≥ 1 x 103 cfu/µg pTF102DNA
Shipping condition: Dry ice
- ig® Thymidine-Auxotrophic EHA101Thy Agrobacterium ElectroCompetent Cells
- DNA (pTF102, 500 pg/µl)
- Recovery medium*
* Client needs to add 50 mg/L of thymidine (no provided) to recovery media before use
- ig® EHA101Thy Agrobacterium ElectroCompetent Cells: -80 °C
- pTF102 control DNA: -20 °C
- Recovery medium: 4 °C
1402 1402-10 1402-30
5×50μl, 15×50μl, 6×100μl, 12×100μl
Aliu E., Azanu MK., Wang K., Lee K. Generation of thymidine auxotrophic Agrobacterium tumefaciens strains for plant transformation https://www.biorxiv.org/content/10.1101/2020.08.21.261941v1.full.pdf
The auxotrophic Agrobacterium tumefaciens strains were developed with support by National Science Foundation Plant Genome Research Program Grant 1725122 and 1917138 to K.W., by the Iowa State University Interdepartmental Plant Biology Major fellowship to EA and MA, by the USDA NIFA Hatch project #IOW04341 and by State of Iowa funds and by the Crop Bioengineering Center of Iowa State University.
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Product Use Limitation and Disclaimers
This product is intended for research purposes only, not for therapeutic or diagnostic purposes in humans or animals. This product contains chemicals which may be harmful if misused or making direct human contact.
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Use this procedure to transform ig® Thymidine-Auxotrophic EHA101Thy Agrobacterium ElectroCompetent Cells. Do not use these cells for chemically transformation.
- Place sterile cuvettes and microcentrifuge tubes on ice.
- Remove competent cells from the -80 °C freezer and thaw completely on wet ice (10-15 minutes).
- Aliquot 1 µl (10pg -1 µg) of DNA to the chilled microcentrifuge tubes on ice.
- 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 pRF102 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.
- 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
- Immediately add 974 µl of Recovery Medium with 50 mg/L of thymidine 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 a 15ml of culture tube.
- Incubate tubes at 30 °C for 3 hours at 200 RPM.
- Spread 20-200 μl cells onto a pre-warmed selective plate. For the pTF102 control DNA, you may plate 100 μl of undiluted transformation mix onto a YT plate containing 50 μg/ml of thymidine, 15 μg/ml rifampicin and 50 μg/ml spectinomycin. Use sterilized spreader or autoclaved ColiRoller™ plating beads to spread evenly.
- 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