Hi all. I´m using calcium-phosphate transfection for 293T. I had never transfected cells with any method. I used two DNA concentrations, 5ug and 10ug per well (6-well plate). I expected lower than optimal results as it was my first time trying the procedure. I obtained about 40% efficiency with 5ug and about 10% with 10ug. My professor says that is inconsistent. I thought perhaps the DNA concentration was too high, as I seem to gather most people use 2.5ug or less, but as an undergrad I don't really know. The plasmid is TurboGFP (SHC003) and had a desirable ratio; and diluted to 5ug/ul as it was highly concentrated. 2xHBS was prepared by myself at 7.11 pH as my professor indicated; after filtering I aliquoted and froze it inmediatelly. I also prepared CaCl2 2.5M; which I also filtered and aliquoted, then kept it at 4°C.
And so I prepared:
5ug GFP: 1 ul GFP (5ug/ul), 8 ul CaCl2, 91 ul molecular grade water.
10ug GFP: 2 ul GFP (5ug/ul), 8 ul CaCl2, 90 ul molecular grade water.
I added each solution dropwise to an equal volume of 2xHBS; this was done under constant vortexing. The solution initially had a yellow-ish colour that went away quickly, and then after a couple minutes it looked white-ish.
I replaced the medium, which I had changed and hour before transfection, with 1 ml DMEM 10%SFB, 1% PenStrep.
I incubated the mixtures 10 minutes and then added it to the cells drowise and trying not to go over the same spot twice. After each drop I shook the plate gently, so the DNA could cover as much of the plate as possible. The medium turned slightly orange. I left this medium for 4 hours.
After the 4 hours I washed the cells with PBS 7 times; as I had seen very big calcium precipitates the only time I had tried it before. Then I replaced with DMEM full. I noticed while washing that some cells were lifting; even though I always take precautions not to treat them roughly.
After 24hrs I took the cells to the cytometer and got the results I already mentioned. I may have done something wrong. My professor expected at least 70% efficiency. I also added 7AAD to check for cell death/damage but I got 0%.
Any tips? Tricks? Thanks to all in advance.
Hi there,
I have performed the transfection using CaCl2 and am sharing my protocol:
To set up a 600 ul reaction in two separate eppendorf (1.2ml total),
Add 10ug of DNA + 30 ul of 2.5M CaCl2 + 300 ul of 2XHBS Buffer + Adjust with water 600 ul. Mix using 1 ml of pipette and let it stand for 10 minutes at Room Temperature. (Total 20ug DNA/6cm dish)
I never changed any media and add the two tubes per 6cm dish drop-wise. Then, wash it after 24 hrs, twice with PBS and add the 10% Media.
Then, I collect the cells after 48 hrs of Transfection.
I have achieved positive 95~100% Efficiency. Also, I never visualized any colour change upon addition.
Hope it helps.
Good Luck.
I would propose a more simple lipofection (e.g. via using Lipofectamine(R) 2000 or 3000).
Pratibha, thank you very much. I think I will try making my solutions again; perhaps something went wrong there. I was told the medium shouldn't stay orange, as you mentioned; so I think new solutions should fix it.
Andreas, we do have Lipofectamine 2000 but we don't have much left, that's why I'm using this method. I think with me being inexperienced maybe there's fear I might waste the Lipofectamine. I should be trying that in the future, but I don't know when.
In general, its your choice and work. Best wishes for your future experiments.
Never tried this method, Why dont use PEI method for transfecting 293T Cells ? Its convenient.
- Badges
- Science topic