Azotobacter Vinelandii

Azotobacter vinelandii

Making electrocompetent cells:

1. Cultivate cells with vigorous shaking at 30 °C to an O.D.₆₂₀ of 0.4-0.5.


2. Harvest by centrifugation at 5,000 rpm for 10 min at 0 °C.


3. Wash with the original culture volume of ice-cold 10% glycerol.


4. Repeat this step three times using a half and a quarter of the original volume, and finally 2-4 ml of the glycerol solution (7.0 x 10⁷ cells/ml final cell concentration).


5. Freeze 40 µl aliquots in liquid nitrogen and store at –70 C.

Electroporation of cells:

1. Add 1 µl (300 ng/µl) plasmid DNA to 40 µl of electrocompetent cells. Homogenize by gently mixing with pipette

several times. Transfer mixture into a prechilled cuvette.

2. Wipe moisture from the cuvette and insert the cuvette into the device.


3. Electroporation:
   
   

Mode	Prokaryotes “O“
Voltage (V)	1,800 V
Time constant (T)	5 ms
No. of pulses (n)	1



4. Immediately add 1 ml AG medium and incubate with shaking at 200 rpm for 1 h at 30 °C.


5. Plate onto selective AG media plates.

Expected results:

Transformation efficiency up to 6.8 x 10⁵ transformants/µg of DNA.