Bacteria Transformation

From FreeBio

{Note: This protocol can be used to introduce a plasmid into TOP10 E. coli cells from Invitrogen.}

  1. Thaw OneShot Top10 Chemically Competent (not electrically competent) cells on ice.
    {Note: Invitrogen TOP10 vials can be found in the -80°C freezer in rm 5088 with purple caps.}
  2. Add 10-15µl cells into each 1.5ml (Eppendorf) tube, pre-chilled on ice.
    {Note: With ligations, have one control for ligation, and one control for the transformation.}
  3. Add 2µl DNA to tube such that the DNA is ~10-20% of final volume.
    {Note: For transformation control, use pUC19 DNA from Invitrogen. Do NOT vortex or pipet up and down!}
  4. Chill on ice for 5-10 min.
  5. Heat shock @ 42°C for 30 sec.
  6. Incubate on ice 2 min.
  7. Add 170µl SOC medium (Invitrogen) to each tube.
  8. Shake @ 37°C for 15 min.
  9. Pipet or pour the transformants onto suitable selective LB plates. Spread cells with glass spreader or glass beads.
  10. Incubate at 37°C overnight. Transformant colonies should appear within 12hrs.