Description
GoldBio’s GV3101 competent 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 further improves the stability of heterologous gene transcripts by inhibiting RNA silencing of foreign genes. These combined properties make GV3101 competent Agrobacterium cells an ideal choice for cDNA or gDNA library construction and the generation of transgenic plants with desirable traits.
Kit Components
Storage/Handling
This product may be shipped on dry ice. GV3101 (pSoup-P19) Agrobacterium chemically competent 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) chemically competent Agrobacterium: 50 µl
- DNA (pCAMBIA1391z Control, 10 ng/µl): 5 µl
- Recovery medium: 1 ml
Antibiotic Selection
Table 1: Antibiotic disc sensitivity for GoldBio’s GV3101 Agrobacterium strains (using standard BD antibiotic discs)
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Antibiotic Selection
|
|
Amp
|
Carb
|
Chlor
|
Gent
|
Kan
|
Rif
|
Spect
|
Strep
|
Tet
|
|
GV3101
|
I
|
R
|
R
|
PR
|
R
|
S
|
R
|
S
|
R
|
S
|
|
GV3101
(pSoup)
|
I
|
R
|
R
|
PR
|
R
|
S
|
R
|
S
|
R
|
R
|
|
GV3101
(pSoup-P19)
|
I
|
R
|
R
|
PR
|
R
|
S
|
R
|
S
|
R
|
R
|
|
S = Sensitive
R = Resistant
R/S = intermediate zones using standard discs.
I = growth in inhibitory zone with standard disc. “Opaque”, not clear zone of inhibition.
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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 ≥3 x 103 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
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