Dandekar & Chandler:Electroporation with pseudomonas or burkholderia: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 20: | Line 20: | ||
* 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 | ||
* Efficiency of electrocompetently-prepared cells '''reduces drastically''' if frozen and used another day -- for best results make cells fresh each time | * Efficiency of electrocompetently-prepared cells '''reduces drastically''' if frozen and used another day -- for best results make cells fresh each time | ||
* Most referenced in the lab, with a nice and detailed protocol: [http://www. | * Most referenced in the lab, with a nice and detailed protocol: [http://www.ncbi.nlm.nih.gov/pubmed/17406227/ mini-Tn7 insertion in bacteria with single ''att''Tn7 sites: example ''Pseudomonas aeruginosa''] | ||
Latest revision as of 13:53, 22 July 2016
Back to Protocols
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
- Most referenced in the lab, with a nice and detailed protocol: mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa
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
- Grow up an overnight culture of your strain in LB.
- Spin down 1 ml of culture (3 min at 8,000 rpm)
- Resupend cells in 1 ml of sterile 10% sucrose, spin again
- Remove sucrose, repeat sucrose wash
- 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.
- 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
- Back dilute 1 mL overnight culture into 100 mL LB; grow to OD600 ~0.5
- Chill cell flask and empty 50 mL conical tubes on ice for 20 minutes; divide cells into tubes
- 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
- Pour off supernatant, wash with 100 mL cold sterile water and pellet
- Pour off super and wash again with 100 mL sterile water; pellet
- Pour off super and wash with 2 mL cold 10% glycerol; pellet
- Pour off super and re-suspend pellet in 200 μL 10% glycerol
- Use 100 μL aliquots for electroporation; add plasmid to cells before transferring to pre-chilled electroporation cuvettes
- Electroporate on setting Ec2 for 2 mm gap cuvettes, or Ec1 for 1 mm cuvettes
- Add 1 ml LB, recover 1 hr at 37 degrees, plate on selective medium