AGL1 chemically Agrobacterium Express Competent Cells

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  • Model: CHC00070
  • 98 Units in Stock
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AGL1 chemically Competent Cells

Product specifications

AGL1                          10 × 100 μl

pCAMBIA2301(control vector,10ng/μl ) 10μl

Store at  –80°C for  12 Month

 

Genotype

C58 RecA (rifR/carbR) Ti pTiBo542DT-DNA Succinamopine

 

Product Description

AGL1 strain is a C58, RecA type background, and the nuclear gene contains a screening label, the rifampin resistance gene RIF and the carboxybenzylpenicillin resistance gene carb. In order to facilitate the transformation, the strain carries a succinalkali Ti plasmid pTiBo542DT-DNA without its own transport function, which contains the VIR gene (VIR gene is a T-DNA inserted plant base. " Because of the necessary components of the group, the T-DNA transfer function of pTiBo542DT-DNA plasmid is destroyed, but it can help transfer the dual vector T-DNA smoothly. The AGL1 strain is suitable for transgenic plants such as rice, Arabidopsis and poplar. AGL1 chemical transformation competent cells produced by our company are produced by special technology. The pCAMBIA2301 was used to detect the conversion efficiency of >104 cfu/ mu g DNA.

 

Method of operation

1. Please store AGL1 Chemically Competent Cell in -80°C for late use. Place it on Room temperature to partial melting.Then Thaw a tube of AGL1 Chemically Competent Cell(100μl )on 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 ℃ 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

AGL1

S

S

R

S

R

EHA105

S

S

R

S

S

LBA4404

S

R

R

S

S

AGL1

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!

 

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