Lipofectamine Plus

COMPONENT VOLUME (PER WELL) X # WELLS = TOTAL VOLUME
Tube 1          
Plasmid: 1 l X _____ = _____
SFM:   99 L  X _____ = _____
TOTAL 100 ml X _____ = _____
Plus Reagent 15 ml X _____ = _____

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COMPONENT VOLUME (PER WELL) X # WELLS = TOTAL VOLUME
Tube 2          
SFM:   92 l X _____ = _____
Lipofectamine 8 L  X _____ = _____
TOTAL 100ml X _____ = _____

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Proceedure:

1.     Day before transfection, plate 150K cells per well of a 6-well plate

2.     Grow overnight

3.     Next day, dilute DNA (0.5g/l) in Serum- and antibiotic-free media in a 15ml tube

4.     MIX

5.     Add Plus Reagent and MIX

6.     Incubate at room temperature for 15 min.

7.     Dilute Lipofectamine in Serum- and antibiotic-free media in a different 15ml tube

Mix gently

8.     Combine Lipofectamine and DNA

Pipette in and out 5-times

9.     Incubate 15 min at room temperature

During:

            Aspirate media and wash 1 X DPBS

            Add 0.8ml Serum- and antibiotic-free media to each well

10.  Add 200ml of the transfection solution dropwise to each plate

11.  Incubate plates at 37C for 5 hr

12.  Add 1ml growth media

13.  17-24 hours later change rinse 1 X with PBS and add 2ml growth media

14.  17-24 hours later switch to differentiation media

15.  Collect the cells 4-6 days later