In our experiment, I used Qiagen’s QIAEXII Gel Extraction Kit (Cat. No. 20021) to prepare the DNA prior to transfection so that it was clear of bacterial endotoxins. I used the “desalting protocol” since I wasn’t extracting the DNA from a gel, but was simply purifying it after restriction digestion to linearize the DNA. Qiagen recommends using their EndoFree Kit, but also mentions that you can use the QIAfilter to do the same thing. I like the QIAEXII because it allows you to purify as much DNA as you want just by adding more beads to the extraction. You can never have the DNA too pure so doing one of these protocols prior to your transfections is important.
After purifying the plasmids, I determined the concentration of the DNA by running 1 µL on a gel and performing “Band Brightness” analysis to estimate the concentration. For example, if I determined that a band of 1 µg of ë DNA BstE II ladder contains 0.149µg of DNA and see that my plasmid band seems to be about four times as bright, I would then determine that the concentration of my DNA is about 0.6µg/uL.
In our experiment, we were transfecting H-293 cells with RPE65 and another plasmid with LRAT + RPE65. Both plasmids were similar and used G-418 as a selector. I also highly recommend that you predetermine the concentration of G-418 prior to doing the transfection. The concentration varies wildly depending on which cell type you are transfecting. I always shoot for a two week selection.
The PolyFect protocol is pretty self-explanatory. I like to have the cells 80% confluent when doing the transfection and leave the reagent on the cells for 24 hours before switching to selecting media. If I have time, I like to switch to plain 10% FBS after the transfection period because I think that the transfection process is stressful to the cells. By giving the transfected cells 24 hours post transfection to recover before beginning selection, some transfected cells that are stressed out might be more likely to survive. My personal theory is that it may also improve the integration of the DNA into the genome. However, I haven’t bothered to truly test this theory. I’m not sure how harmful the PolyFect reagent is to the cells so I try not to keep the transfection reagent on the cells too much past the point when it is needed. I believe overnight is plenty of time but I often leave it on for 24 hours. The protocol actually says 24-48 hours so I guess that is what you might want to do.
Since the ratio of PolyFect reagent to DNA is a critical factor, you will want to do at least two concentrations of DNA. In my experiment in a 100 mm TC dish, I used 4 µg DNA and 0.4 µg of DNA, leaving the amount of PolyFect constant. Both worked very well but the 4 µg had way more transfections. So at least I can safely recommend that you should use the PolyFect when transfecting H-293cells. I have no complaints.
Later… I may have taken my early success with PolyFect to heart too soon. When attempting to transfect the ARPE-19 cell line using the same constructs, the experiment failed. I noticed a few transfected cells, but the transfectants never divided. Granted, ARPE-19 cells aren’t your model transfection cell type. Currently, I’m attempting the same transfection using Qiagen’s Effectene reagent. It is specifically designed for such pesky cell types. Maybe I’ll let you know how that goes in a later review.
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