Transfection of SF9 best procedure
1- Start from suspension cells (detaching the cells from adherent flasks may cause cell disruption.
2- Count 8x10^5 cells per well and plate them in adherent media (centrifuge them and resuspend them in this media).
3- During centrifugation prepare plating media: 1.5 ml unsupplemented media +10% FBS and 8.5 ml of unsupplemented media.
4- After 15 minutes the cells should be attached. Replace the media with the plating media.
5- While waiting for cells to attach prepare the mixes:
Mix A
Cellfectin 8ul+
Grace’s media unsupplemented 100ul.
Mix B
LR recombination product 10ul
Grace’s media unsupplemnted 100ul
Add B to A and wait for 30 minutes.
Add to the cells dropwise.
The day after replace the media with supplemented +FBS 10%+ Ab/Am+ ganciclovir. Wait at least 72 hours and proceed with testing the cells by western blot.