Transfection with Lipofectamin Plus.
Optimised for transfection of NT2 stem cells
Method prepared by Dr. Anne Nøremølle, University of Copenhagen, Department of Medical Biochemistry and Genetics, Copenhagen, Denmark.
Cell growth
Grow NT2 cells in a big bottle until confluence ≈ 20-60000000 cells
Trypsinate, count and aliquot cells
1,5 x 106 cells per small bottle (in 5 ml DMEM+/+)
Grow until 40 – 80% confluence, 1-2 days
Transfection with Lipofectamin Plus
Per small bottle:
Mix 2 μg plasmid DNA with 8 μl PLUS reagent or miRCURY™ Knockdown probe§
Incubate 15 min, RT
Mix 12 μl Lipofectamin with 125 μl DMEM without serum/including antibiotic*/+glutamax
Add DNA-PLUS mix
Incubate 15 min, RT
Remove medium from cells
Wash x 2 med 4 ml DMEM without serum/without antibiotic/without glutamax
Add 2 ml DMEM without serum/with* antibiotic/+glutamax to the DNA/LipofectaminPLUS
Mix
Add to cells immediately
Incubate cells in 3 hours at 37°C and 5% CO
2
Remove medium from cells
Wash x 2 med 4 ml DMEM without serum/without antibiotic/without glutamax
Add fresh DMEM with serum/with antibiotic/with glutamax
Incubate at 37°C and 5% CO2
*Antibiotic is optional, depending on the experimental setup
§This protocol was made for co-transfection with plasmid DNA. If you do not make co-transfection substitute plasmid DNA with miRCURY™ Knockdown probe in nM range
