Transfection of Sf9/Sf21 Cells
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
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
PO Box B
Frederick, MD 21702
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