Transfection using Calcium Phosphate - (Feb/04/2008 )

Does anybody have experiences in calcium phosphate transfection?
Is the efficacy of this method depends on vector size?

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The main problem is optimizing the reagents. Once you have optimized it, then its the cheapest transfection reagent.

I am not sure of the maximum vector size with this method. But I know people have tried ~ 10-12kb without any problem.

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In fact I can transfect the GFP vector into the cells and I can see the expression through fluorescent microscope. However, the vector (same backbone of the vector expressing GFP) with my insert did not express as I cannot detact it by Western blot.

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did you do a positive transfection control to verify that the transfection procedure was ok.

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Yes, I performed 3 different transfections,
1 is my gene of target,
2 is GFP,
3 is both.

I can always get the GFP band with its size ~27kDa, but not my gene of interest.

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I guess you must have verified the presence of the transgene of interest in the other vector.

Is the protein secreted or may be degraded? What I mean is is there any thing specific for the protein which makes it difficult to detect?

Another thing, is your western blot protocol working? like antibodies for this gene of interest.

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Thank you very much that you always help me. I detect the transgene also by anti-GFP antibodies as it is also GFP tagged (in N terminal). It should not be a problem for expression and detection. I think I will check the sequence of the plasmid again if the Insert is still in or not.

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Oh, I saw they are contaminated 3 days post transfection, when they were still fine a day before ...

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Are you sure its contaminated? did you change media after transfection?

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Yes, I gave the cells 2min glycerol shock (15% Glycerol in HBS), then fresh medium for the following days. The first 36h were still looking fine, but a day after the medium became turbid ...

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