NanoBio: Bacterial Transformation

Traditional Transformation of Chemically Competent Cells

 * 1) Thaw competent cells (One Shot Chemically Competent TOP10 from Invitrogen, catalog # C4040-03) on ice.
 * 2) *Note: if transforming ccdB-containing plasmids, use One Shot ccdB Survival - T1R Chemically Competent Cells (Invitrogen catalog # C7510-03) instead.
 * 3) Place 10-15 µL cells in pre-chilled Eppendorf tubes.
 * 4) Add 2 µL ligated vector or diluted mini-prepped plasmid (1:10 to 1:50 will work), and chill on ice for 30 min.
 * 5) *The volume of plasmid should be 10-20% of the total volume.
 * 6) *Do not pipet or vortex.
 * 7) Heat shock at 42 °C for 30 s.
 * 8) Incubate on ice for 2 min.
 * 9) 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.
 * 10) *If you are in a hurry, you can directly plate Amp resistant plasmids on Amp containing plates.
 * 11) Add all media to a selectable marker LB plate.  Use glass beads to streak the plate.
 * 12) 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.
 * 4) * Plan ahead so that each plasmid has different antibiotic resistance.
 * 5) * If the concentrations of the mini-prepped plasmids are not similar, you may want to dilute so that the added plasmids are equinormal.
 * 6) * You may also need to up concentrations of the miniprepped plasmids if they are particularly large (>10kb)
 * 7) *The volume of plasmid should be 10-20% of the total volume.
 * 8) Chill on ice for 10 min.
 * 9) *Do not pipet or vortex.
 * 10) Heat shock at 42 °C for 40 s.
 * 11) Incubate on ice for 2 min.
 * 12) Add 200 µL SOC medium (~10 x volume; Invitrogen), and shake at 37 °C for 1 hour.
 * 13) Add all media to a LB plates with both appropriate antibiotics. Use glass beads to streak the plate.
 * 14) Incubate at 37 °C.  Transformants should appear within 16 hrs.
 * 15) * 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.


 * Heather M. Jensen 19:52, 11 June 2008 (CST):

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