Fugene transfections - (Oct/11/2012 )
I think I must be really stupid but my effeciencies with calcium phosphate actually work better than fugene/lipofectamine!! What is a typical protocol for fugene transfection ? does adding fugene to the diluent with dna make a difference than making separate dilutions of dna and fugene in serum free medium and mixing ? also what is the typical complexing time ? what efficiency should i expect for an endothelial cell line (p9)? any tips and suggestions welcome
Fugene in the presence of water forms small charged droplets called lipospheres with the DNA inside them due to charges on the DNA that are not present in the medium/water (medium is mostly water). You should be adding the fugene directly to the DNA/diluent mix.
Complexing should be 15-45 min, but can be longer.
Can't comment on the endothelial line though.