NanoBio: Bacterial Transformation

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Traditional 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.
  • CAjoF 19:52, 29 January 2008 (CST):

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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