GV3101 (pSoup-P19) Agrobacterium Electrocompetent Cells

GoldBio’s GV3101 electrocompetent Agrobacterium cells are a highly efficient
tool for genetic transformation of dicotyledonous and monocotyledonous
plant species, such as Arabidopsis thaliana, tobacco, potato, and corn.
These cells carry the nopaline-type Ti plasmid pMP90 (pTiC58DT-DNA) that
contains the vir genes required for the transfer and integration of
T-DNA into the plant genome. Additionally, the cells are resistant to
rifampicin, gentamicin, and tetracycline, which allows for selection of
transformed cells.

The pSoup plasmid carried by the cells is required for the
replication of pGreen, 62SK, and pGs2 series plasmids. The p19 protein
is derived from tomato bush dwarf virus and improves the stability of
heterologous gene transcripts by inhibiting RNA silencing of foreign
genes. These combined properties make GV3101 electrocompetent
Agrobacterium
cells an ideal choice for cDNA or gDNA library construction and the
generation of transgenic plants with desirable traits.

Table 1: Antibiotic disc sensitivity for GoldBio’s GV3101 Agrobacterium strains (using standard BD antibiotic discs)

Product Specifications

Competent cell type: ElectroCompetent
Species: A. tumefaciens
Strain: GV3101 (pSoup-P19)
Transformation efficiency: ≥1 x 10⁸ cfu/µg pCAMBIA1391z DNA
Blue/white screening: No

Storage/Handling: This product may be shipped on dry ice. GV3101 (pSoup-P19) 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

• GV3101 (pSoup-P19) ElectroCompetent Agrobacterium: 50 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 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.

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