Lonza Amaxa
Lonza Amaxa kdorfman Fri, 12/06/2019 - 14:34Nucleofection 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