Nucleofection

Nucleofection with Lonza Amaxa

Order VCA-1005 Cell Line Nucleofector Kit L (25 reactions $347)

Transfection Protocol

  • Warm medium in MatTek dishes to equilibrate CO2
  • Warm additional 0.5 mL medium in sterile microfuge tube
  • Make mucleofection solution, RT, per flask:
    • 19 µL supplement 1
    • 81 µL reagent L for LLCPk (Kit R for 3t3, HeLa)
  • Remove medium from flask
  • Rinse with PBS
  • Add trypsin
  • Incubate ~5 min, or until cells are floating
  • Add cold medium
  • Transfer to sterile 15 mL centrifuge tube
  • Spin 10 min, slow speed
  • Remove medium. Pellet should be soft
  • Loosen pellet by flicking tube
  • Add all 100 µL nucleofection solution plus the DNA (one of the following):
    • 2 µg plasmid DNA
    • 5 µg BAC
    • 5 µL siRNA (20 µM)
  • Pipet up and down to mix
  • Transfer to nucleofection cuvette
  • Insert into nucleofector device, choose program
    • X001 for LLCPK
    • I-013 for HeLa
    • U-030 for 3t3
  • Resuspend cells in 500 µL warm medium
  • Distribute in drops on coverslips of MatTek dishes