A4 Agrobacterium Strain

A4 Agrobacterium Strain

STR3001    Packing  100ul

Store  at  -80℃


A4 Agrobacterium Strain Information

Resistance: Kan
Culture medium: TY
Strain category: Agrobacterium rhizogenes
Culture conditions: 30℃, aerobic, TY
Preservation method: 20% glycerin, -80℃
Basic application: Transgene
 

A4 Agrobacterium Strain Genotype

Agrobacterium rhizogenes(kan^R) Ar A4 Ri(agropine type)

A4 Agrobacterium Strain Description

A4 Agrobacterium tumefaciens is a natural Agrobacterium alkaline strain. It has kanamycin resistance and can be cultured on YT medium. The optimal temperature for culture is 30 ℃.Rhizobium is a Gram negative soil bacterium belonging to the genus Agrobacterium in the Rhizobiaceae family. It can infect most dicotyledonous plants, a few monocotyledonous plants, and a few gymnosperms. The Ar.A4 Agrobacterium tumefaciens strain contains the Agrobacterium tumefaciens Ri plasmid, which has a wide range of hosts (corn, tobacco, carrots, licorice, etc.) and is resistant to kanamycin.

Operating instructions:

1. This product contains a portion of glycerin bacteria. When using this glycerin bacteria, you can dip a small amount of coated plate or liquid culture on the surface of glycerin bacteria without complete melting. It can also be used after complete thawing, but with the increase of freeze-thaw times, the activity of bacteria will gradually decline.

2. In order to ensure the purity of bacteria and avoid other bacterial contamination, we should first draw the plate, and then select the monoclone colony for subsequent operation.

 

Antibiotics for Agrobacterium

LB and YEB 

TY formula (1L)
Tryptone 5g
Yeast extract 3g

1. Add water to a volume of 1L, dissolve completely, and sterilize at 121 degrees Celsius for 20 minutes
2. Prepare a 1M calcium chloride aqueous solution and sterilize it at 121 degrees Celsius for 20 minutes at high temperature
3. Add 10ml of sterile 1M calcium chloride aqueous solution to every 1L of sterilized TY liquid nutrient solution.

*If preparing TY solid culture medium, add 15g agar powder.




Electroporation of Agrobacterium tumefaciens

1.1 Preparation of Electrocompetent Cells
See Lin (1995) for additional information.

1. Inoculate 1.5 L of YM broth in a 2.8 L Fermbach flask with an aliquot from log phase culture of A. tmefaciens.
2. Incubate at 30°C overnight, shaking at 300 rpm to a density of 5-10x 107 cells/ml.
3. Decant the cells into sterile 500 ml centrifuge bottles and pellet the cells by centrifugation at 3000 x g for 10 min at 4℃.
4. Carefully pour off and discard the supematant, place the centrifuge bottles with the cell pellets on ice.
5. Add ~50 ml of sterile, ice-cold 10% glycerol to each of the bottles and vortex toresuspend the cell pellets; bring the volume in each of the centrifuge bottles to 500 ml with sterile, ice-cold 10% glycerol.Pellet the cells by centrifugation at 3000 x g for 10 min at 4℃; pour off and discard the supermatant.
6. Wash the cells again as in step 5.
7. Resuspend each of the cell pellets in 5 ml of sterile, ice-cold 10% glycerol and transfer to a chilled 30 ml Oakridge tube.Pellet the cells by centrifugation at 3000 x g for 5 min at 4℃; pour off and discard the supernatant.
8. Resuspend the cell pellet in 0.5 ml of sterile, ice-cold 1 M sorbitol; the final cell volume should be ~1.5 ml and the cell concentration should be ~ 5 x 1010 cells/ml. Dispense 200 ul aliquots of the electrocompetent cells in sterile 1.5 ml microfuge tubes; freeze thecells in an isopropanol-dry ice bath, then store at -70 ℃. The cells are stable for about6 months under these conditions.

1.2 Electroporation

1. Pipette the DNA samples (up to 5 ul) to be electroporated into sterile 1.5 ml microfugetubes; the DNA should be in either water or TE.Place tubes on ice.
2. For each DNA sample to be electroporated, add 1 ml of YM broth to a 17 x 100 tube at room temperature, and place a 0.1 cm electroporation cuvette on ice.
3. Thaw the electrocompetent A. tumefaciens cells on ice.For each DNA sample to be electroporated, add 20 ul of electrocompetent cells to each DNA sample; gently tap thetubes to mix.
4. Set the MicroPulser to "Agr". See Section 4 for operating instructions.
5. Transfer the DNA—cell samples to the electroporation cuvettes and tap the suspension
   to the bottom of the tube.Place the cuvette in the chamber slide.Push the slide into thechamber until the cuvette is seated between the contacts in the base of the chamber.Pulse once.
6. Remove the cuvette from the chamber and immediately use the YM broth in the17x 100 mm tube to transfer the cells from the cuvette to the tube.
7. Check and record the pulse parameters. The time constant should be about 5 milliseconds.The field strength can be calculated as actual volts (kV) / cuvette gap (cm).
8. Incubate the cells 3 hr at 30 °C, shaking at 250 rpm. Plate aliquots of the electroporated cells on YM agar plates containing the appropriate selective media. Incubate plates for 48 hrs at 30°C.

1.3 Solutions and Reagents For Electroporation

1. YM broth:

0.4 g yeast extract,
10 g mannitol,
0.1 g NaCl,
0.1 g Mgso4,
0.5 g K2HPO4.3H20,
dissolve in 1.0L of water and adjust to pH 7.0. Autoclave.For YMplates,add 15 g agar/1 L of YM broth.


Caution:

1. This product is FOR RESEARCH USE ONLY!

2. This product is glycerol packed, Please kindly culture it as soon as possible then harvest and store at -80℃.

3.  Shipping temperature is 2-8℃.

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