Transformation of chemically competent bacterial cells

Goal:

To introduce plasmids into a strain of bacteria

Contributor: Heather Jensen

Important Notes:

  • Only plasmids up to ~12kB can be introduced into chemically competent bacteria. If the plasmid(s) you wish to introduce are larger than this, use the electroporation procedure.

Single Transformation of Chemically Competent Cells

  1. Thaw competent cells (One Shot Chemically Competent TOP10 from Invitrogen, catalog # C4040-03) on ice.
    • Note: if transforming ccdB-containing plasmids, use One Shot ccdB Survival – T1R Chemically Competent Cells (Invitrogen catalog # C7510-03) instead.
    • Note: if transforming a plasmid containing streptomycin resistance as a selection marker, use Mach 1 cells instead (TOP10 cells are natively strep-resistant).
  2. Place 10-15 µL cells in pre-chilled Eppendorf tubes.
  3. Add 2 µL ligated vector or diluted mini-prepped plasmid (1:10 to 1:50 will work), and chill on ice for 30 min.
    • The volume of plasmid should be 10-20% of the total volume.
    • Do not pipet or vortex.
  4. Heat shock at 42 °C for 30 s.
  5. Incubate on ice for 2 min.
  6. Add 170 µL SOC medium (~10 x volume; Invitrogen), and shake at 37 °C for 15 min for Amp selection, or 1 hr for Chl, Kan, or Tet selection.
    • If you are in a hurry, you can directly plate Amp resistant plasmids on Amp containing plates.
  7. Add all media to a selectable marker LB plate. Use glass beads to streak the plate.
  8. Incubate at 37 °C. Transformants should appear within 16 hrs.

Simultaneous Transformation of Chemically Competent Cells

Note: this protocol is for a different line of competent cells than described above.

  1. Thaw competent cells (Ultra BL21(DE#) Competent Cells from Edge BioSystems) on ice.
  2. Place 10-15 µL cells in pre-chilled Eppendorf tubes.
  3. Add 1.0 µL of each 1:10 diluted mini-prepped plasmid.
    • Plan ahead so that each plasmid has different antibiotic resistance.
    • If the concentrations of the mini-prepped plasmids are not similar, you may want to dilute so that the added plasmids are equinormal.
    • You may also need to up concentrations of the miniprepped plasmids if they are particularly large (>10kb)
    • The volume of plasmid should be 10-20% of the total volume.
  4. Chill on ice for 10 min.
    • Do not pipet or vortex.
  5. Heat shock at 42 °C for 40 s.
  6. Incubate on ice for 2 min.
  7. Add 200 µL SOC medium (~10 x volume; Invitrogen), and shake at 37 °C for 1 hour.
  8. Add all media to a LB plates with both appropriate antibiotics. Use glass beads to streak the plate.
  9. Incubate at 37 °C. Transformants should appear within 16 hrs.
    • It is ideal to also plate out each plasmid separately on appropriate selector LB plates to confirm that each plasmid may transform into this particular line of cells at the selected concentration.

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