Transformation of Competent Cells

Materials

  • Water bath warmed to 42°C
  • Agar plates with appropriate antibiotic, cooled to room temperature
  • SOC media

Competent Cell Choice

  • NEB 5-alpha (NEB, # C2987I) for HiFi Assemblies
  • JM109 (Promega, # L2001) for cloning in pGEM-T Easy Vector (Promega, # A1360)
  • DH5-alpha (made in-house) for other purposes (e.g., C. elegans Fire Lab plasmids)

Note: Follow the kit protocols for commercial cell transformation.

Protocol (JM109 and DH5-alpha)

  1. Take competent cells out of -80°C and thaw on ice.

  2. Add 1 - 5 μL of plasmid (usually 10 pg - 100 ng) into ~100 μL of competent cells in a cold 1.5 mL tube. Gently mix by flicking.

  3. Place the competent cell and DNA mixture on ice for 20-30 min.

  4. Heat-shock in a 42°C water bath for 30-60 s. (45 s. is best). Do Not Shake.

  5. Place on ice for 2 min.

  6. Add 900 μL of SOC.

  7. Incubate at 37°C with shaking (225 RPM) for 45 min.

  8. Plate bacteria on agar plates, typically 3 plates with 100 uL, 200 uL, and 300 uL each. Pipette bacteria onto the plate, add ~10 glass beads, put the lid on the plate and slowly roll the beads through the liquid and around the entire surface area of the plate.

  9. Dump used beads into a glass bottle with 100% ethanol for later re-use.

  10. Incubate the plates at 37°C overnight.