Dandekar & Chandler:Electroporation with pseudomonas or burkholderia

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(Electroporating pseudomonas the quick and dirty way)
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==General Info About Electroporating Pseudomonas or Burkholderia==
==General Info About Electroporating Pseudomonas or Burkholderia==
* Electroporation with non-''E. coli'' strains, ''P. aeruginosa'' and ''B. cenocepacia'' in particular, can be hit or miss
* Electroporation with non-''E. coli'' strains, ''P. aeruginosa'' and ''B. cenocepacia'' in particular, can be hit or miss

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General Info About Electroporating Pseudomonas or Burkholderia

  • Electroporation with non-E. coli strains, P. aeruginosa and B. cenocepacia in particular, can be hit or miss
  • Efficiency of electrocompetently-prepared cells reduces drastically if frozen and used another day -- for best results make cells fresh each time


Electroporating pseudomonas the quick and dirty way

from Brook

  • Note: This only works well for replicating plasmids. If you are trying to get in a suicide plasmid, then you are better off using conjugation - Brook
  • Makes one aliquot
  1. Grow up an overnight culture of your strain in LB.
  2. Spin down 1 ml of culture (3 min at 8,000 rpm)
  3. Resupend cells in 1 ml of sterile 10% sucrose, spin again
  4. Remove sucrose, repeat sucrose wash
  5. Resupend cell pellet in 100 ul of 10% sucrose, add DNA (1 ul of a high copy plasmid mini prep works fine), electroporate on setting Ec2 for 2 mm gap cuvettes, or Ec1 for 1 mm cuvettes.
  6. Add 1 ml LB, recover 1 hr at 37 degrees, plate on selective medium

Electroporating burkholderia the quick and dirty way

from Thao

  • Note: This only works consistently for B. thailandensis
  • Makes 1-2 aliquots
  • Inoculate your cells into LB early in your day in case doubling is slow
  • Turn on the centrifuge early to make sure it is at 4°C when you are ready to spin
  1. Back dilute 1 mL overnight culture into 100 mL LB; grow to OD600 ~0.5
  2. Chill cell flask and empty 50 mL conical tubes on ice for 20 minutes; divide cells into tubes
  3. In centrifuge pre-chilled to 4°C, pellet cells for 20 min (this is way excessive -- I do 5 min - Nicole) at 5,000 rpm
  4. Pour off supernatant, wash with 100 mL cold sterile water and pellet
  5. Pour off super and wash again with 100 mL sterile water; pellet
  6. Pour off super and wash with 2 mL cold 10% glycerol; pellet
  7. Pour off super and re-suspend pellet in 200 μL 10% glycerol
  8. Use 100 μL aliquots for electroporation; add plasmid to cells before transferring to pre-chilled electroporation cuvettes
  9. Electroporate on setting Ec2 for 2 mm gap cuvettes, or Ec1 for 1 mm cuvettes
  10. Add 1 ml LB, recover 1 hr at 37 degrees, plate on selective medium
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