Transfection of Sf9/Sf21 Cells

Greg Tobin tobin at fcs260c.ncifcrf.gov
Mon Jun 6 12:42:59 EST 1994


With respect to transfection of Sf-9 and Sf-21 cells:

Yes, electroporation has been used to co-transfect viral DNA targets with
recombination vectors, but why go through the trouble?  Calcium phosphate
co-precipitation and lipofectin work fine.  BTW, DOTAP seems not to work.

Calcium phosphate transfection:
1.	Allow 2,000,000 cells to attach to the surface of T25 flasks.
2.	Mix gently 1 ug AcNMPV (or linearized BacPAK) DNA with 2 ug
	transfer vector in 0.75 ml transfection buffer (25 mM HEPES
	pH 7.1, 140 mM NaCl, 125 mM CaCl2 - note that pH is critical).
3.	Replace the media from flasks with 0.75 ml Grace's media (with 
	10% FCS but not supplemented with lactalbumin hydrolysate and
	yeastolate).
4.	While gently rocking the flasks, slowly add the DNA solution
	to the media.
5.	After a 4 h incubation, remove DNA solution, rinse cells, and add
	5 ml complete TNM-FH media.  Incubate 3-5 days.

In my experience with the BacPAK system, you will want to set up plaque 
assays using dilutions of 1:10, 1:100, and 1:1000 if you use Lipofectin
and Neat, 1:10, and 1:100 if you use calcium phosphate.


-- 
Greg Tobin, Ph.D.                        tobin at lcms-1.ncifcrf.gov
LCMS 
PO Box B
Frederick, MD 21702



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