Electro comp. A.t

14/07/10

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  1. Production of electro competent Agrobacterium tumefaciens cells
  2. Electroporation of Agrobacterium tumefaciens

 

Production of electro competent Agrobacterium tumefaciens cells

Perform the following steps under sterile conditions

  1. Inoculate 5 ml LB + Rifampicillin (10 µg/ml) and incubate over night at 28 oC with 150 rpm

  2. Inoculate 2 x 300 ml preheated LB medium + Rifampicillin (10 µg/ml) with 1 ml overnight culture

  3. Incubate at 28 oC with 150 rpm for app. 24 hours until OD600 = 0.5

  4. Cool the cultures on ice for 15 minutes

  5. Pellet cells by centrifugation 4500 rpm (Sorvall RC5) at 4 oC.

  6. Discard the supernatant and resuspend pellet in 50 ml ice cold sterile MilliQ water

  7. Pellet the cells and discard the supernatant

  8. Wash the cells in 10% glycerol for 1 hour

  9. Pellet the cells and discard the supernatant

  10. Resuspend the cells in 3 ml 10% glycerol

  11. Aliquot the resuspended cells in 100 ml portions and store at – 80 oC.

 

 

Electroporation of Agrobacterium tumefaciens

Aim:              To introduce a vector into Agrobacterium tumefaciens

Materials:   

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    50 µl electrocompetent cells (typically LBA4404)

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    500 µl SOC medium

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    2 x (LB + selection) plates

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    Electroporation cuvette (0.2 mm electrode gap)

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    Electroporation apparatus: Bio-Rad Gene Pulser II

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    Your vector DNA (typically 1 µl of Miniprep)

Procedure

  1. Place the electroporation cuvette on ice
  2. Get the competent cells from the – 70 oC freezer
  3. Thaw the cells on ice
  4. Switch on the electroporation apparatus and change the settings to the following

Voltage = 2.50 kV (push “set voltage” and then “Raise”)

Capacitance = 25 mF

Resistance = 200 W

  1. Place 1 µl of your DNA (Miniprep) on the internal side to the cuvette
  2. Gently use the 50 µl of competent cells to flush the DNA containing droplet to the bottom of the cuvette. Gently thump the cuvette to the table a couple of times to remove air bubbles
  3. Dry the exterior sides of the cuvette with a paper towel (to prevent short circuits)
  4. Place the cuvette in the apparatus
  5. Push in the two red buttons simultaneously and hold them in until you hear a beep. (If the sample explodes then see below*).
  6. Remove the cuvette from the apparatus and apply 450 µl SOC medium as quickly as possible (fast application of SOC increases the number of surviving cells)
  7. Pour the cells into a new 1.5 ml Eppendorf tube
  8. Incubate the cells at 28 oC for 1 ˝ hour with 350 rpm.
  9. Plate the cells onto two selective plates (1/10 and 9/10 of the volume)
  10. Incubate the plates at 28 oC for 2-3 days
  1. Clean the used cuvettes: first rinse with 70 % ethanol, then with water and then 96 % ethanol. Air dry the cuvette in the UV box to degrade any remaining DNA.

 

* If the salt concentration in the DNA sample is too high the electric shock will be discharged very fast resulting in a small explosion, giving a loud “CLICK" or "BANG”. Repeat the procedure but with 0.5 µl of your Miniprep, as the competent cells are killed in the “explosion”.

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Dette sted blev sidst opdateret 14. July 2010