Cloning and expression analysis of Agrobacterium mediated electropositive cells

EHA105(pSoup) Electrocompetent Cell

Agrobacterium mediated electroporation of competent cells

Product label:

EHA105 strain; Agrobacterium competent cells; Rifampicin (Rif) and rifampicin; Kanamycin (kan) kanamycin; EHA101； LBA4404； GV3101； AGL1； Plant genetic modification research;

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The EHA105 strain is modified from the EHA101 strain, with a chromosome background of C58 and a nuclear gene containing a screening tag - rif resistance gene. In order to facilitate transformation operations, this strain carries a non self transporting succinylated Ti plasmid pEHA105 (pTiBo542DT DNA), which contains the vir gene (the vir gene is an essential element for T-DNA insertion into the plant genome, and although the T-DNA transfer function of the pEHA105 (pTiBo542DT DNA) plasmid itself is disrupted, it can assist in the smooth transfer of the transferred binary vector T-DNA). Transferring the help plasmid pSoup into the EHA105 strain yields the EHA105 (pSoup) strain, which can assist in the replication of pGreen, 62SK, and pGs2 series plasmids in Agrobacterium, while endowing the strain with tetracycline (tetR) resistance, making it suitable for transgenic operations in plants such as rice and tobacco.

The EHA105 (pSoup) electropositive cells provided by our company (Maokang Biotechnology) are specially processed and suitable for the transformation of large plasmids. The genotype is C58 (rifR) Ti pEHA105 (pSoup) (pTiBo542DT DNA) Sucinamopine (pSoup tetR), and the transformation efficiency is>105 cfu/μ g DNA according to pGs2 plasmid detection.

Product components:

Storage and transportation conditions:

Storage: -80 ℃ storage, valid for 1 year.

Transportation: Dry ice transportation.

Precautions

1) Sensory cells should be stored at -80 ℃ and should not be repeatedly frozen or stored for too long to avoid reducing the conversion efficiency of competent cells.

2) The entire conversion process is strictly carried out according to the corresponding temperature and sterile conditions requirements.

3) The volume of plasmid added should not exceed 1/10 of the receptive volume. Impurity of plasmids or contamination with organic substances such as ethanol can lead to a sharp decrease in transformation efficiency. Doubling the plasmid results in a decrease of one order of magnitude in transformation efficiency.

4) To ensure the highest conversion efficiency, the entire operation process should be as gentle as possible and kept at a low temperature.

5) The conversion of high concentration plasmids can correspondingly reduce the final coating amount.

6) When the clone density on the tablet is too high, due to insufficient nutrition, clone growth slows down and bacterial colonies become smaller. To obtain large colonies, the amount of plasmid used should be reduced.

7) The purpose of adding rifampicin to the culture medium is to prevent the growth of miscellaneous bacteria and screen for Agrobacterium; Adding streptomycin or qingda meisu based on the resistance of the strain used can prevent the loss of Ti plasmids, but streptomycin is not conducive to the transgenic operation of Agrobacterium. Generally, streptomycin or qingda meisu is not considered when cultivating Agrobacterium because the probability of Ti plasmid loss is extremely low (basically negligible)

8) For your safety and health, please wear lab coats and disposable gloves when operating.

Instructions for use

1) Take out the 0.1 cm electric shock cup and cup lid from the storage solution and invert them on clean absorbent paper for 5 minutes. Let them drain the water and stand upright for 5 minutes to allow the ethanol to evaporate completely. Immediately insert them into ice and compact the ice surface. The top of the electrode cup should be 0.5 cm away from the ice surface to facilitate the lid. Let it stand in the ice for 5 minutes to fully cool it down.

2) Take competent cells stored at -80 ℃ and insert them into ice for 5 minutes until they melt.

3) Add 0.01~1 μ g (volume not exceeding 6 μ l) plasmid DNA to 50 μ l of competent cell suspension, mix well by hand tapping the bottom of the tube, and immediately insert into ice. Gently blow and mix the competent cells plasmids with a gun, then quickly transfer them to an electric shock cup, cover the cup lid, and keep the empty tube for later use. [Note: Due to the high efficiency of competent transformation, use Zuihao once for a preliminary experiment to determine the amount of plasmid used in Zuija].

4) Start the electrospinning device and set the parameters: C=25 μ F, PC=200 ohm, V=2400 V (refer to Bio rad for this parameter, please refer to the recommended steps of the electrospinning device for specific use). Quickly place the electric shock cup into the electrospinning slot and insert it into the ice quickly after the electric shock is completed.

5) Add 700 μ l of antibiotic free LB and transfer it to a competent empty tube. Shake and culture at 28 ℃ for 2-3 hours.

6) Centrifuge at 6000rpm for 1 minute, take about 100ul of supernatant, and then gently blow the resuspended bacterial cells with a gun. Then add it to LB or YEB plates containing the corresponding antibiotics, and gently spread the cells evenly with a sterile glass rod. Let it stand at room temperature for 10 minutes. After the liquid is absorbed, invert the plate and incubate at 28 ℃ for 2-3 days. Note: When the tablet only contains the dual carrier antibiotics used for transformation, it can be cultured at 28 ℃ for 48 hours; When adding dual carrier antibiotics to the plate and then adding 10 μ g/ml rifampicin, it needs to be cultured at 28 ℃ for 48-60 hours; when adding dual carrier antibiotics to the plate and then adding 20 μ g/ml rifampicin, it needs to be cultured at 28 ℃ for 60-72 hours; it is not recommended to use rifampicin exceeding 20 μ g/ml for plate or liquid culture.

7) Liquid culture for cloning purposes:

J small shake culture: Add 1ml of LB liquid medium containing the corresponding antibiotic to a 15ml round bottomed breathable test tube, select 1-2 single colonies from freshly cultured plates for inoculation, shake at 30 ℃ and 200rpm for 24-48 hours.

K Large shaking of bacteria: Add less than 20ml of LB liquid culture medium containing corresponding antibiotics into a 100ml breathable triangular flask, take small shaking bacterial solution and inoculate it at a ratio of 2%, shake at 30 ℃ and 200rpm for 24-48 hours.

【 Attention 】: For Agrobacterium (aerobic bacteria), both solid agar plates and liquid shaking require a large amount of oxygen. The key to successful liquid shaking of bacteria is to ensure that the culture medium contains sufficient dissolved oxygen, which can be controlled by the following methods: using breathable test tubes or triangular flasks; Ensure that the nutrient solution has a large cross-section and a small thickness; Antibiotics should be added as little or no as possible, and antibiotics that must be added should be used at low concentrations.

Related products 【 Sensory cell series 】

Appendix 1: Formulas for Solid and Liquid LB Culture Media

Appendix 2 Solid and Liquid YEB Medium Formulas

Appendix 3: General Table of Antibiotic Sensitivity of Common Agrobacterium

——Written/Edited by V. Shallan [Copyright belongs to MKBio Maokang]

Shanghai Maokang Biotechnology Co., Ltd. is a company engaged in the research of reagents, instruments, laboratory consumables, and experimental services in the fields of life sciences and biotechnology. It mainly engages in cell biology, botany, molecular biology, immunology, biochemistry, and proteomics. In the fields of biopharmaceuticals and diagnostic reagent research and production. Our company adheres to the business philosophy of "people-oriented, honesty and trustworthiness, and contract keeping". Adhere to the principle of 'quality assurance' to provide high-quality products to our customers.