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Actinomyces viscosus

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.501 04/2002 Microorganism Actinomyces viscosus Cell type Bacteria, gram negative Molecules injected Plasmid DNA (in 1 mM Tris-HCl-0.1 mM EDTA (pH 8.0)) Growth medium Lactobacillus carrying medium (LCM) with 20 mM DL-threonine Washing solution Sterile, distilled water; sterile 10% glycerol Electroporation solution Sterile, 10% glycerol Outgrowth medium Complex medium (CAM) Cuvette 2 mm gap width Reference Yeung, M. K. and Kozelsky, C. S. 1994 Journal of Bacteriology 176. No. 13 4173-4176 Making electrocompetent cells:
  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.
Electroporation of cells:
  1. Thaw bacterial suspensions on ice. Add up to 5 l (100 ng) plasmid DNA to 100 l (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/g of DNA.



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