Actinomyces Viscosus

Actinomyces viscosus

1. Grow a 500 ml cell culture in the early exponential phase until an O.D.⁶⁰⁰ of 0.2 is reached. Chill on ice for 1.5 h.
   
2. 2. Harvest by centrifugation.
   
3. Wash extensively first with a large volume of ice-cold sterile distilled water and then with sterile 10% glycerol.
   
4. Resuspend in 10% glycerol to a final volume of 2 ml. Store in aliquots at –80 °C.

1. Thaw bacterial suspensions on ice. Add up to 5 µl (100 ng) plasmid DNA to 100 µl (5 x 10⁸) of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into prechilled cuvette.
   
2. Wipe moisture from the cuvette and insert the cuvette into the device.
   
3. Electroporation:
   
   

   Mode	Prokaryotes “O“
   Voltage (V)	2,500 V
   Time constant (T)	5 ms

   
   
4. Dilute cells with 1 ml of CAM and incubate for 2 h at 37 °C.
   
5. Plate onto selective brain heart infusion agar plates; incubate at 37 °C for up to 5 days.

Expected results:

Transformation efficiency up to 2.9 x 10⁷ transformants/µg of DNA.