EHA101 感受态细胞(化转)/ EHA101 chemi

cally Agrobacterium Express Competent Cells

价格￥980

品牌LSM Bio
货号CHC00073
更新日期2025年07月16日

武汉维克赛思科技有限公司

7 年钻石会员

EHA101 chemically Competent Cells

Product specifications

EHA101 10 × 100 μl

pK7WGF2锛坈ontrol vector锛?0ng/μl ) 10μl

Store at –80°C for 12 Month

Genotype

C58 (rifR) Ti pEHA101 (pTiBo542 D T-DNA) (kanR) Nopaline

Product Description

EHA101 strain of C58 background, containing screening label, rifampicin resistant gene RIF in nuclear genes, in order to facilitate the transformation of the operation, the strains carrying nopaline type Ti plasmid pEHA101 without its transport function (pTiBo542DT-DNA), the plasmid containing vir gene (VIR T-DNA gene was inserted into the plant genome essential elements (pTiBo542DT-DNA, pEHA101) the plasmid T-DNA transfer function is damaged, but can be transferred to the binary vector T-DNA help smooth transfer). PEHA101 (pTiBo542DT-DNA) Ti plasmid contains screening Tags: Kan, which endows EHA101 and kanamycin resistance, and is suitable for transgenic operation of plants such as maize, rice and tobacco. The EHA101 chemically transformed cells produced by our company were made by a special process. The pK7WGF2 plasmid (resistance to splendin) was used to detect the conversion efficiency of >104 cfu/ mu g DNA.

Method of operation

1. Please store EHA101 Chemically Competent Cell in -80°C for late use. Place it on Room temperature to partial melting.Then Thaw a tube of EHA101 Chemically Competent Cell锛?00μl 锛塷n ice.

2. Add 0.01-1µl plasmid DNA to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. .Place the mixture on ice for 5 minutes,Do not mix. Place the mixture on Liquid Nitrogen for 5 minutes, Do not mix. Place it in the 37°C water,then immediately transfer the tube back into ice for 5min. Do not mix.

3. Add 700ul LB or YEB liquid nutrient medium. then put on the Constant temperature shaker for 2-3 hours.

4. Pellet the mixture by centrifugation at 6000rpm/min for 1min. Pour off about 100ul of cell supernatant, then resuspend the cell pellet in the remaining medium by gently vortexing the tube. Spread the cell on the LB or YEB Resistance plates by antibiotic, then inverted and Incubate 2-3days at 28-30°C. (When the plate contains only 50 μg / ml kan, 28 ° C for 48 h; in the plate at the same time by adding 50 μg / ml kan, 20 μg / ml rif, need 28 鈩?for 60 h; if the plate contains 50 Μg / ml rif requires 28 ° C for 72-90 h).

Antibiotics for Agrobacterium

Antibiotics	Method	Stock solution	fluid
Carb	Dissolved in DDW(Double distilled water), filtration sterilization by 0.22 m membrane filtration	50mg/ml	50ug/ml
Kan	Dissolved in DDW(Double distilled water), filtration sterilization by 0.22 m membrane filtration	50mg/ml	50ug/ml
Strep	Dissolved in DDW(Double distilled water), filtration sterilization by 0.22 m membrane filtration	10mg/ml	50ug/ml
Rif	Dissolved in DMSO, filtration sterilization by 0.22 m membrane filtration	10mg/ml	20ug/ml
gent	Dissolved in DDW(Double distilled water), filtration sterilization by 0.22 m membrane filtration	20mg/ml	40ug/ml

Resistance of the Agrobacterium (R Resistance; S Sensitive)

Strain	carb	Strep	Rif	Gent	kan
AGL-1	R	S	R	S	S
EHA101	S	S	R	S	R
EHA105	S	S	R	S	S
LBA4404	S	R	R	S	S
EHA101	S	S	R	R	S

LB and YEB

Component	LB (Liquid)/L	LB( Solid /L	Component	YEB (Liquid)/L	YEB ( Solid /L
Tryptone	10g	10g	Tryptone	5g	5g
Yeast extract	5g	5g	Yeast extract	1g	1g
NaCl	10g	10g	Beef extract	5g	5g
NaOH	PH TO 7	PH TO 7	Sucrose	5g	5g
Agar	-	15g	MgSO₄*7H₂O	0.49g	0.49g
			NaOH	PH TO 7	PH TO 7
			Agar	-	15g

Caution

1. When the plasmid is added, the volume should not be more than 1/10 of the volume of the competent cells.The plasmid is impure or has organic pollution such as ethanol. The transformation efficiency is drastically decreased. The plasmid is doubled and the conversion efficiency is decreased by an order of magnitude.

2. Mix the plasmid with gentle operation. Transformation of high concentrations of plasmid can reduce the amount of bacteria used in the final coating.

3. On the plate when the positive clone density is too large, due to lack of nutrition, positive clonal growth slow, colony smaller, in order to obtain large colonies, should reduce the amount of plasmid.

4. Rifampicin concentration should not be higher than 25 μg / ml, too high rifampicin concentration is not conducive to Agrobacterium growth, will reduce its growth rate and conversion efficiency. The plate used to calculate the conversion efficiency of the company contains only 50 μg / ml kan, and the conversion efficiency is reduced to 1/2 if the plate contains 20 μg / ml rif.

5. The addition of rifampicin in the culture medium is to prevent the growth of bacteria and screen Agrobacterium. According to the resistance of the strain, the addition of the Ti plasmid can prevent the loss of Ti plasmid, but the screening of antibiotics by Ti plasmid is not beneficial to the transgenic operation of Agrobacterium, So the general cultivation of Agrobacterium does not consider these antibiotics, Ti plasmid loss probability is very low (can be ignored).

6.STORAGE AND HANDLING: Competent cells should be stored at–80°C. Storage at –20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above –80°C, even if they do not thaw.

7. This product is FOR RESEARCH USE ONLY!

Related Agrobacterium Products

Agrobacterium

A4 Agrobacterium Strain