Retroviral calcium phosphate transfection - How much DNA should I use?? (Jul/10/2008 )
I am trying to transfect A293T cells to produce retrovirus to infect human neuroblastoma 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 an MSCV GFP plasmid into the cells, and I have been cotransfecting with pEQPAM3e (gag-pol) 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.
Also, does anyone have anyone have any experience using chloroquine or a glycerol shock in transfections?
Any input is greatly appreciated.
Thanks so much!
P.S. Any transfection tips for A293T or Phoenix cell lines, using CaPO4, Mirus, lipofectamine, or whatever, are warmly welcome as well.
I would go for a commercial kit for transfection. The "gold standard" in our lab is Fugene HD (or formerly Fugene6). We have good experiences for several cell lines, including A293T.
It has very high trfx efficiencies but the lowest cytotoxicity and off-side effects (compared to other commercial kits like lipof.).
Don't spend too much time, effort and costs for consumables with Ca-Phosphat. It is really old-fashioned, not very reproduceble and has only low efficiency - save it for buying the trfx reagent. AND they have a trial pack in some countries available
Once you have the kit do the optimization titration (ratio of trfx reagent to DNA) like described in the pack insert and use the optimized ratio.... it's really worth it.
More information under www.powerful-transfection.com
Wish you much success!
Maybe you can give feedback how you decided and how it worked out.