Gama:Competent bacteria transformation

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Competent bacteria transformation and plasmid DNA amplification

Materials

  • Competent bacteria DH4α
  • Plasmid DNA at concentration above 200μg/mL
  • Antibiotics recommended working concentrations:

Ampicilin – 100 µg/mL

Kanamycin – 30 100 µg/mL

Procedure

  • Make sure your competent bacteria stock is held at -80°C and take a sample for transformation (all bacteria handlings should be performed on ice; use the Bunsen burner to guarantee sterile conditions).
  • Pipette 10 µL of bacteria to a new eppendorf tube.
  • Add 0,5 µL of plasmid DNA and keep the mixture on ice for 15 minutes.
  • Perform a thermal shock by putting your tubes to a dry bath heated at 42°C during 45 seconds.
  • Keep the mixture on ice for two or more minutes.
  • Add 50 µL of LB liquid media (bacteria and DNA volumes can be changed according with transformation efficiency; in those cases, add a volume of LB which is five times larger than the previous tube volume).
  • Let your transformations incubate at 37°C with shaking for 45 minutes.
  • Plate your mixtures in a Petri dish full of LB agar solid growth media. The Petri dish must be previously prepared by adding the growth media and the antibiotic corresponding to plasmid encoded resistance gene (the antibiotics concentration depends on which one we are using).
  • Let your bacterial cultures grow overnight at 37°C.
  • In the next morning, select a colony from your dish.
  • Prepare a 50 mL Falcon tube with 5 mL of LB liquid growth media and antibiotic (once more, the antibiotic volume depends on which we are using and of its stock solution concentration).
  • With a pipette tip, pick the selected colony and inoculate it in the Falcon tube.
  • Let the cultures grow overnight at 37°C with shaking.
  • In the following morning, perform a plasmid DNA purification procedure
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