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Salmonella typhimurium

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.532 01/2002 Microorganism Salmonella typhimurium LT2 Cell type Bacteria, gram negative Molecules injected Plasmid DNA (pBR322 in TE buffer) Growth medium LB medium Washing solution 10 mM HEPES, pH 7.0; 10% glycerol Electroporation solution 10% glycerol Outgrowth medium SOC medium (without antibiotics) Cuvette 2 mm gap width Reference Binotto J., et al 1991 Canadian Journal of Microbiology 37 474-477 Making electrocompetent cells:

1. Incoculate a flask of LB 1:100 with a fresh overnight culture, grow at 37 C with shaking to an O.D.640 of 0.75. Chill the cells in an ice-water bath for 15 min. 2. Harvest by centrifugation (4,000 x g, 10 min., 4 C). 3. Wash one time in the original culture volume with HEPES and one time with 10% glycerol using 1/100 of the original volume. 4. Resuspend in 10% glycerol.

Electroporation of cells:

  1. Add 2 l plasmid DNA (20 pg to 20 ng) to 40 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) 2,400 V Time constant (T) 5 ms
  4. Immediately add 1 ml SOC medium and transfer to a sterile culture tube. Incubate for 1 hour at 37 C with shaking.
  5. Dilute the cells in SOC medium and plate on selective LB plates.
Expected Results: Transformation efficiency up to 4 x 108 transformants/g of DNA.



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