Bacteria

scientificprotocols authored over 3 years ago

Authors: Chenzhong Kuang 

Material and Reagents

  1. SOC
    • 2% Tryptone
    • 0.5% Yeast Extract
    • 10mM NaCl
    • 10mM MgSO4
    • 10mM MgCl2
  2. 1.5 mL microfuge tubes
  3. 42° C waterbath
  4. Ice
  5. 37° C shaker

Protocol

  1. Thaw competent cells on ice. 20–200µL per tube
  2. Add max. 20µL of a ligation reaction
  3. Mix very gently!
  4. Incubate the tubes on ice for 30 min
  5. Heat shock the cells for 45 sec to 2 min at 42°C
  6. Place the tubes immediately on ice for at least 2 min
  7. Add 800µL of SOC medium to each tube
  8. Incubate for 1 hour at 37°C and shake vigorously
  9. Spin down briefly and remove most supernatants
  10. Resuspend cell pellet with the rest SOC medium in the tube by pipetting
  11. Plate out the suspension on a LB agar plate containing the appropriate antibiotic.
  12. Incubate the plates overnight at 37°C

Remarks

  1. I have use this protocol to transform DH5 alpha competent cells successfully
  2. When transforming purified plasmid into competent cells, I just add 1µL plasmid DNA solution. Then plate out only 10–20µL bacterial suspension to the plate instead of all
  3. Efficiency depends on ligation reaction and competent activity.

DOI

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