PlatE Transfection using ExGen500

[Luke]

Plat E Transfection using ExGen 500

1.   Maintain PlatE cells in DMEM-10 with HEPES, NaPyruvate, NEAA, PS, 1ug/mL puromycin and 10ug/mL blasticidin.
2.   24 hours before transfection, plate ~2-3 million cells per 100mm dish in complete media
3.   Remove media and add 3mL fresh DMEM-10
4.   In each eppendorf tube combine 5ug DNA, 300uL 150mM NaCl, mix gently
5.   Add 16.45 uL ExGen 500, mix for 10sec, incubate 10 minutes RT
6.   Add mixture on top of cells dropwise, rock back and forth to spread out
7.   Incubate O/N 37? C
8.   Remove supernatant and add 3mL fresh media, incubate O/N 37? C
9.   Harvest supernatant (now at 48hours post transfection)
10.   Add fresh media, incubate O/N 37? C
11.   Harvest supernatant at 72 hours post transfections
12.   Filter supernatant by 0.45uM filter, freeze.