EHA105 (pSoup) Agrobacterium ElectroCompetent Cells

EHA105 (pSoup) ElectroCompetent Agrobacterium cells are designed to provide high transformation efficiencies for applications such as cDNA or gDNA library construction. The strain contains a rifampicin resistance gene (rif) and an amber basic Ti plasmid pEHA105 (pTiBo542DT-DNA), which carries the vir gene necessary for the insertion of T-DNA into the plant genome. Although the pEHA105 plasmid is incapable of transferring its own T-DNA, it can still transfer foreign binary vector T-DNA.

The presence of the pSoup helper plasmid is required for the replication of pGreen, 62SK, and pGs2 series plasmids, and also helps with Agrobacterium-mediated transformation of dicotyledonous plants like Arabidopsis thaliana, tobacco, and potato, as well as monocotyledonous plants such as corn.

GoldBio’s EHA105 Agrobacterium strain was generated, and primary clone supplied by Dr. Elizabeth Hood.

Kit Components

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

Competent cell type: ElectroCompetent
Species: A. tumefaciens
Strain: EHA105 (pSoup)
Transformation efficiency: ≥4 x 10⁸ cfu/µg pCAMBIA1391z DNA
Blue/white screening: No

Storage/Handling: This product may be shipped on dry ice. EHA105 (pSoup) 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

• ≥1.6 x 10⁸ cfu/µg efficiency with electroporation.

Reagents Needed for One Reaction

• EHA105 (pSoup) ElectroCompetent Agrobacterium: 25 µL
• DNA (pCAMBIA1391z Control, 500 pg/µL): 1 µL
• Recovery medium: 1 mL

Table 1: Antibiotic disc sensitivity for GoldBio’s EHA105 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 ≥4 x 10⁸ 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