Agrobacterium ElectroCompetent Cell Combo Pack

Product Description

GoldBio’s Agrobacterium ElectroCompetent Combo Pack is perfect for scientists who require multiple agrobacterium competent cell strains for their research.The Agrobacterium ElectroCompetent Combo Pack contains three, 50 µl aliquots, of each: GV3101, AGL1, EHA105 and LBA4404 strains, along with pCambia and our proprietary Agro Recovery Media.

GV3101 Strain (3 x 50 µl): Contains a C58 chromosomal background with rifampicin resistance and the Ti plasmid pMP90 (pTiC58DT-DNA) with gentamicin resistance. GV3101 is also resistant to Chloramphenicol and this antibiotic cannot be used for selection. The GV3101 Ti plasmid has the T-DNA region sequences deleted and transformation with a binary vector containing the missing T-region results in a functional T-DNA binary system that allows for transfer of genetic material into a host plant’s genome. Therefore, this system is often used for Agrobacterium-mediated transformation of several dicots such as Arabidopsis thaliana, tobacco, potato, and monocots like corn.

AGL1 Strain (3 x 50 µl): Contains a C58 chromosomal background that carries an insertion mutation in its recA recombination gene which stabilizes recombinant plasmids. It also carries rifampicin and carbenicillin resistance in its genome for selection. AGL1 contains the Ti plasmid pTiBO542 from which the T-DNA region sequences have been deleted. Transformation with a binary vector containing the missing T-region results in a functional T-DNA binary system that allows for transfer of genetic material into a host plant’s genome. Therefore, this system is often used for Agrobacterium-mediated transformation of Arabidopsis thaliana as well as maize and other monocots.

LBA4404 Strain (3 x 50 µl): Useful for transgenic operations of tomatoes, tobacco and other plants.LBA4404 contains a rifampicin resistance gene (rif) and also has streptomycin resistance.LBA4404 strain also contains a octoprine-type Ti plasmid pAL4404 without self-transport function, which contains the vir gene.

EHA105 Strain (3 x 50 µl): 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.

Table 1: Antibiotic selection for GoldBio’s Agrobacterium strains

Antibiotic Selection

Amp

Carb

Chlor

Gent

Kan

Rif

Spect

Strep

Tet

100
µg/ml

100
µg/ml

30
µg/ml

100
µg/ml

30
µg/ml

50
µg/ml

25
µg/ml

50
µg/ml

50
µg/ml

50
µg/ml

GV3101

S

S

R

PR

R

S

R

PR

R

S

EHA 105

S

S

R

R

R

S

R

R

R

S

LBA 4404

S

S

S

n/a

S

S

R

S

R

S

AGL-1

PS

R

S

n/a

S

S

R

S

R

S

C58C1

S

S

S

n/a

S

S

R

S

R

S

S = Sensitive
R = Resistant
PS = Partial Sensitive (Some growth, but no colonies)
PR = Partial Resistance (Small colonies or some growth in concentrated areas)


Product Specifications
Competent cell type: ElectroCompetent
Species: A. tumefaciens
Format: Tubes
Blue/white screening: No

Storage/Handling: This product may be shipped on dry ice. 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.

Reagents Needed for One Reaction

  • ElectroCompetent Agrobacterium: 25 µl
  • DNA (pCAMBIA1391z, 500 pg/µl): 1 µl
  • Recovery medium: 1 ml

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 ≥1 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

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