AGL-1 Agrobacterium Electrocompetent Cells

GoldBio’s AGL-1 Agrobacterium Electrocompetent Cells allow you to obtain high transformation efficiency in applications such as gDNA or cDNA library construction. Our AGL-1 strain harbors the C58 chromosomal backbone with an insertion mutation in its recA recombination gene. This mutation stabilizes recombinant plasmids. AGL-1 also has rifampicin and carbenicillin resistance genes in the genome useful for selection. A functional T-DNA binary system can be built using our AGL-1 strains as the T-DNA region has been deleted in the Ti plasmid pTiBO542 and instead it has a binary vector containing the missing T-region. The binary system makes possible to transfer genetic material into a host plant’s genome. Therefore, our system is often used for Agrobacterium-mediated transformation in mono and dicotyledonous species such as Arabidopsis thaliana, maize, and other plants.

Kit Components

• Competent Cells
• 1 x 12 mL Recovery Media
• 1 x 10 µL Control Plasmid (pCAMBIA1391z Control, 500 pg/µL)

Product Specifications
Competent cell type: ElectroCompetent
Species: A. tumefaciens
Strain: AGL-1
Transformation efficiency: ≥5 x 10⁷ cfu/µg pCAMBIA1391z DNA
Blue/white screening: No

Storage/Handling: This product may be shipped on dry ice. AGL-1 Agrobacterium Electrocompetent cells should be stored at -80°C, pCAMBIA1391z Control DNA should be stored at -20°C and recovery medium should be stored at 4°C immediately upon arrival. When stored under the recommended conditions and handled correctly, these products should be stable for at least 1 year from the date of receipt.

Genomic Features

• ≥5 x 10⁷ cfu/µg efficiency with electroporation.

Reagents Needed for One Reaction

• AGL-1 ElectroCompetent Agrobacterium: 25 µL
• DNA (pCAMBIA1391z Control, 500 pg/µL): 1 µL
• Recovery medium: 1 mL

Table 1: Antibiotic disc sensitivity for GoldBio’s Agrobacterium strains (using standard BD antibiotic discs)

Quality Control
Transformation efficiency is tested by using the pCAMBIA1391z control DNA supplied with the kit and using the protocol given below. Transformation efficiency should be ≥5 x 10⁷ CFU/µg pCAMBIA1391z DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.

General Guidelines

• 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 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 1 µg of plasmid into a given volume of competent cells.

• TE = Colonies/µg/Dilution
  • Colonies = the number of colonies counted
  • µg = amount of DNA transformed in µg
  • Dilution = total dilution of the DNA before plating

Example: Transform 1 µl of (10 pg/µl) control plasmid into 25 µl of cells, add 975 µl of Recovery Medium. Dilute 10 µl of this in 990 µl of Recovery Medium and plate 50 µl. Count the colonies on the plate the next day. If you count 250 colonies, the TE is calculated as follows:

Colonies = 250
µg of DNA = 0.00001
Dilution = 10/1000 x 50/1000 = 0.0005
TE = 250/0.00001/0.0005 = 5.0 × 10¹⁰