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Actinobacillus pleuropneumoniae

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.539 04/2002 Microorganism Actinobacillus pleuropneumoniae Cell type Bacteria, gram negative Molecules injected Plasmid DNA (in water) Growth medium Columbia broth with 1% IsoVitaleX and 10 g/ml -NAD Washing solution 15% glycerine Electroporation solution 15% glycerine Outgrowth medium Columbia broth with 1% IsoVitaleX and 10 g/ml -NAD Cuvette 2 mm gap width Reference Frey, J. 1992 Research in Microbiology 143 263-269

Making electrocompetent cells:

1. Grow cells to mid-exponential growth phase at an O.D.650of 0.5.
2. Harvest by centrifugation at 3,000 x g for 10 min at 4 C.
3. Wash twice in 15% glycerine at 4 C.
4. Resuspend in 1/20 volume of 15% glycerine and keep at 4 C.

Electroporation of cells:

1. Add 3 l (300 g/ml) plasmid DNA to 125 l 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) 1,250 V Time constant (T) 5 ms
4. Immediately add 1 ml outgrowth medium and incubate for 3 h.
5. Plate onto selective Columbia agar plates.

Expected results:

Transformation efficiency up to 1 x 107 transformants/g of DNA.



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