CaPO4 Transfection - How much DNA should I use?? (Jul/10/2008 )
I am trying to transfect A293T cells to produce retrovirus to infect human tumor cells. My associate researcher and I (an intern) have done 3 transfections so far using the Mirus TransIT reagent, with results ranging from 0.9%-17.4% transfection efficiency. We're not working with a clonal cell line, so those numbers are too low and run the risk of selecting a specific subset of cells not representative of the tumor as a whole.
My PI has asked me to try calcium phosphate transfection, so earlier in the week I prepared 8 different batches of HBS w/pH ranging from 6.6- 8.0. To optimize the transficiency, I will first be trying to insert MSCV GFP plasmid into the cells, and I have been cotransfecting with pEQPAM3e and pSRalphaG packaging envelopes.
My question is this, how much DNA do I use? How much plasmid vs. env? I've found protocols that say anywhere from 10 ng- 30 ng, and none of them specify what type/what amount, if any, of helper vector DNA is necessary. Perhaps this is a rookie's question, but the research associate I work with is on vacation for two weeks, and no one else in the lab has done CaPO4 for years.
Any suggestions?
Thanks so much!
P.S. Any transfection tips for A293T or Phoenix cell lines are greatly appreciated! 
I don't want you to get no answer at all.. But it's been a while since I've done it.
The ratios that we use in the lab are 2:2:3 (ug) and that's GagPol:VSVG:clone you want to use.
Tips... Our HBSS was always around pH 7 and worked well.
Since you've obviously have done it before, I won't point out the obvious... Don't squirt media on the cells and such
But one thing that might help. We always used chloroquine (and I'm not sure how much, sorry!) to permeabiilize the cells more.
Hope it helps...
Good luck!
The ratios that we use in the lab are 2:2:3 (ug) and that's GagPol:VSVG:clone you want to use.
Tips... Our HBSS was always around pH 7 and worked well.
Since you've obviously have done it before, I won't point out the obvious... Don't squirt media on the cells and such
But one thing that might help. We always used chloroquine (and I'm not sure how much, sorry!) to permeabiilize the cells more.
Hope it helps...
Good luck!
Thank you so much! That is very helpful. I had come across a protocol mentioning chloroquine but wasn't sure whether or not to use it because none of the others said anything about it.
Along those lines, does anyone have any opinions regarding a glycerol shock?
Hi,
We are using chloroquine - 6 ul of 25 mM chloroquine in 6 ml of DMEM. It is added right after the calcium phosphate-DNA precipitate and the media is changed to DMEM 7 hours after the transfection. Chloroquine inhibits lysosomal DNases by neutralizing vesicle pH, however it is slightly toxic, therefore it should not be left on the cells longer than 7-8 hours.
I have also tried glycerol shock on 293Ts - 2-16 hours after the transfection. The media was removed and 2 ml of 10% glycerol in DMEM was added to the cells. Following 3 min incubation at room temperature, the glycerol solution was removed, the cells were washed twice with PBS and the media was replaced with DMEM. The best efficiency of transfection was obtained when the glycerol shock was conducted 2 hrs after the transfection.