EHA105 Chemically Competent Agrobacterium

The EHA105 strain is useful for transgenic operations of rice, tobacco and other plants.

Description

Intact Genomics EHA105 Chemically Competent Agrobacterium cells are optimized for the highest transformation efficiencies which is ideal for applications requiring high transformation efficiencies, such as with cDNA or gDNA library construction. The EHA105 strain is useful for transgenic operations of rice, tobacco and other plants. EHA105 contains a rifampicin resistance gene (rif). EHA105 strain also contains an amber basic Ti plasmid pEHA105 (pTiBo542DT-DNA) without self-transport function, which contains the vir gene.

Specifications
Competent cell type:              Chemically Competent
Species:                                    A. tumefaciens
Strain:                                      EHA105
Format:                                    Tubes
Transformation efficiency:       ≥ 1 x 10⁵  cfu/µg pCAMBIA1391z  DNA
Blue/white screening:              No
Shipping condition:                  Dry ice

Reagents Included

• EHA105 Chemically Competent Agrobacterium
• DNA (pCAMBIA1391z, 500 pg/µl)
• Recovery medium

Note: Liquid nitrogen is required.

Storage
EHA105 Chemically Competent Agrobacterium:  -80 ºC
pCAMBIA1391z control DNA:   -20 ºC
Recovery medium:   4 ºC

Quality Control
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.

General Guidelines

Follow these guidelines when using EHA105 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) pCAMBIA1391z 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 kanamycin-selection.