Calcium phosphate transfection - media turns pink and cloudy

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Best
Best's picture
Calcium phosphate transfection - media turns pink and cloudy

I transfected 293T with calcium phosphate kit from clontech. as soon as i add the transfection mix the medium turned cloudy and pink and a lot of black stuff floating around. the cells were almost fried the next day. this time the 293Ts were in McCoy's 5A medium. The one previous time when I did this, the cells were in DMEM when this problem did not occur.
are there  anythoughts on this?

Thanks

Sami Tuomivaara
Sami Tuomivaara's picture
Best,

Best,

As you probably know the precipitation is expected, and necessary for successful transfection... Trying to avoid some media with calcium phosphate transfection has also been known, see here () and here.

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EDIT: References to the first 3 links:
1st link: Jordan M et al. (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Research 24: 596–601.

2nd link:

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Steelman LS et al. (?) Elucidation of Signal Transduction Pathways
by Transfection of Cells with Modified Oncogenes. Methods in Molecular Biology, Vol. 218: 203-220.

3rd link:

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Jones JS & Risser R (1993) Cell Fusion Induced by the Murine Leukemia Virus Envelope Glycoprotein. Journal of Virology 67: 67-74.

I looked up the media compositions and what strikes my eye is that McCoy's 5A has lot more phosphate salt than DMEM. This may cause lot of things, for example, too fast precipitation that makes the crystals too big or too small and DNA doesn't enter the cell at all. Also, there can be some phosphate toxicity to the cells.

Cheers,

Best
Best's picture
Thank you so much Suola. It

Thank you so much Suola. It is good to know that the medium matters. I was able to access the 2nd link and not the first.