Description
GoldBio’s EHA105 (pSoup-P19) chemically competent Agrobacterium cells are optimized for high transformation efficiencies, making them ideal for applications such as cDNA or gDNA library construction. These cells are resistant to rifampicin, chloramphenicol, and streptomycin, providing versatility in selection and antibiotic resistance EHA105 strain contains a rifampicin resistance gene (rif) and an amber basic Ti plasmid pEHA105 (pTiBo542DT-DNA) without self-transport function, containing the vir gene.
The pSoup plasmid present in these cells is required for the replication of pGreen, 62SK, and pGs2 series plasmids, increasing the range of applications for the EHA105 (pSoup) system. 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. These competent cells are suitable for Agrobacterium-mediated transformation of dicots such as Arabidopsis thaliana, tobacco, potato, as well as monocots like corn, making them an ideal choice for plant molecular biology research.
GoldBio’s EHA105 Agrobacteriumstrain was generated, and primary clone supplied by Dr. Elizabeth Hood.
Kit Components
Storage/Handling
This product may be shipped on dry ice. EHA105 (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
- EHA105 (pSoup-P19) Chemical 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 EHA105 Agrobacterium strains (using standard BD antibiotic discs)
| 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 | 5
µg/ml | 50
µg/ml | 50
µg/ml | 5
µg/ml |
| EHA105 | R | R/S | R | n/a
| R/S | S | R | S
| R | S |
| EHA105
(pSoup)
| R | R/S | R | n/a | R/S | S | R | S | R | R |
| EHA105
(pSoup-P19) | R | R/S | R | n/a
| R/S | 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. |
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 ≥2 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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