Can someone share a protocol for inexpensive and efficient transfection of H1299 cells? I would like to transfect these cells in 15cm dishes with DNA plasmids. Lipofectamine 2000 works quite well but it is expensive for large scale applications. I tried also amaxa nucleofection with a selfmade Phosphate buffer, which is also working but the survival rate is bad. Maybe someone has a custom buffer formulation and optimized amaxa settings for these cells.
I would also recommend you try PEI. I have not worked with H1299 cells, but I developed a protocol for PEI transfection of 293T cells that gives around 100% transfection-efficiency (as good or better than Fugene/lipo). It costs around 1/5000th the price of Lipo2000, so is virtually free. If you contact me I can send the protocol and you can give it a try. It's no more difficult than using Lipo2000.
I also don't know if this will work in your cell line but calcium phosphate is an inexpensive transfection technique.
http://www.flemingtonlab.com/Protocols/CalciumPhosphateTransf.pdf
Arjan and Simon, thanks to both of you for your answers. I already tried linear and branched PEI, but none of both worked so far. The linear form was cytotoxic already at 2:1 (PEI:DNA) and the branched form did not transfect the cells. I also tried the Ca-phosphate method but it was not efficient, but I should try the protocol that Simon shared, it is a bit different from mine.
Have you tried electroporation?
It has a high transfection efficiency for a number of cell lines.
Herinder, no until now I did not use conventional electroporation, I only used nucleofection (amaxa electroporation).
Conventional electroporation does not require special buffers.
You zap the cells in growth medium.
In our hands lipofectamine LTX with Plus reagent works very well, protocol according to the instructions of the manufacturer
I used lipofectamine 2000, the efficiency is very high but definitely not inexpensive. One other method could be calcium-phosphate transfection.
hi, I guess you must select either of the existing protocols using calcium phosphate, lipofectamine or electroporation, where the last one is most efficient and no need to have the machine if some lab around can help you.
I agree with Jacopo that lipofectamine 2000 is efficient in that case. Nano liposome now appeared as new approach for that purpose now.
I can suggest one lipofection reagent called DreamFect Gold which is quite good for Lung carcinoma or the Magnetofection method.
If you have a friendly colleague down the hall with an Amaxa system (or the Bio-rad one... cannot remember the name), you could pop it in with one of those, then expand. I've found that their tested protocols for cell lines are reliable [especially for DNA]. If you factor in labor and time, you will probably end up spending as much optimizing one of the cheaper reagents.
Of course, if labor and time are not a concern, then as others have already noted optimizing calcium phosphate is going to be dirt cheap in the face of everything else.
Thanks to all of you. I'll try the Calcium Phosphate method once more, first time it didn't work, but perhaps there was something messed up. As i mentioned, Lipofectamine 2000 is working very well but it is rather expensive if you plan to transfect 30 15 cm dishes or so...
I heard calcium phosphate works best with other cell lines. Have you considered using PEI? It is quite cheap and less tricky than lipo. It will not have the same efficiency as lipo, but to optimize it, it is best to have at least 70%confluency, and PEI:DNA ratio of 5:2. Same protocol as lipo but you can leave transection mix with opti overnight.
I would also recommend you try PEI. I have not worked with H1299 cells, but I developed a protocol for PEI transfection of 293T cells that gives around 100% transfection-efficiency (as good or better than Fugene/lipo). It costs around 1/5000th the price of Lipo2000, so is virtually free. If you contact me I can send the protocol and you can give it a try. It's no more difficult than using Lipo2000.
Jet-PEI is far more efficient and less toxic than lipofectamine. Yes, it's much cheaper too!
Hi,
I am using CaCl2 to transfect HeLa and Hek 293T cells, and it is working beautifully. You just need to optimize pH of HBS buffer which is used during procedure. You need a minute quantity of DNA to get high efficiency of transfection,plus,it is super cheap.
I have also used TurboFect from Fermentas and it worked beautifully for many different cell lines. It was even better than the electroporation for some endothelial cell lines. And it is quite cheap and efficient. If You use CaCl2 method it is better to use commercialy available reagents than prepare them themselves as the method is very, very sensitive to not even errors but just small deviations from the right pH and/or concentration. So if Your pH-meter is not freshly calibrated by the person who knows how to do that properly and the scale is not very sensitive (and maybe not clean enough) the small deviations will kill the efficiency.
Hi all, can anybody suggest me optimum amount of DNA (eg. pegfp) to be used for electroporation of h1299 cells (around 500000 cells)?
Hi Dietmar, I recommend DreamFect Gold or Ecotransfect as a cheap way to transfect this cells. Otherwise there is also the technology called Magnetofection. Le t me know if you want samples. best regards
If you want to large scale transfections and use a nucleofection protocol for H1299 cells, I am happy with X-001 and solution T of the Amaxa kit. I use 4-20 ug plasmid (4 ug if your plasmid is toxic to the cells) for a 80% confluent 15 cm dish and have an efficiency of 85% with GFP and a good survival. I never quantified survival, but there are hardly any floaters and the cells flourish well. If I need lots of cells with the same transfection in different plates, I usually transfect 3X15 cm plates with 3 nucleofection reactions that I collect in a falcon tube and then split over 24 10cm plates, which I will harvest at about 75% confluency 48 hrs later.
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