Lonza Amaxa

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