query about efficacy of calcium phosphate transfection - (Feb/29/2008 )

Hi,

I AM CONDUCTING TRANSFECTION ON COS-CELLS USING CALCIUM PHOSPHATE TRANSFECTION METHOD ACCORDING TO FLAMINGTON'S LAB PROTOCOL. HOWEVER, THE TRANSFECTION EFFICIENCY IS VERY LOW AND AM NOT ABLE TO GET A GOOD RECEPTOR EXPRESSION LEVEL. ANY IMPROVISATION OR TIPS ON THE SAME WILL BE APPRECIATED.

(I USE MEDIA WITHOUT ANTIBIOTICS AND USUALLY KEEP THE PLATES FOR 2 DAYS AFTER TRANSFECTION)

THANKS

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Nowadays fewer people use CaPO4 method than lipid based reagents. Check the pH of your stock solutions. It has to be very close to what is listed in the protocol.

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Some cells are easier to transfect than others, eg 293 are very easy with calcium phosphate.

Check indeed the pH of your 2xHBS as that's the most crucial factor. Have you tried with a plasmid encoding a fluorescent protein to check you efficiency?

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...nevertheless in some cases Ca-phosphate is to prefer, f.i. in the case of cytotoxic proteins (which do not kill but transform the cell)

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Hi,

Thanks for your reply. In fact, I have prepared my stock, aliquoted and frozen down . I just thaw out the required amount. I know CaPo4 is not used much but lipofectamine and stuff is too expensive as I have to transfect a lot and that consumes loads of the reagent. Can you suggest some other efficient techniques? Thanks a lot

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Hi, Thanks for your reply.

Ya i think my Ph is alright. I have also transfected COS-7 cells with GFP construct and it looked alright about 60-70% efficiency. I still cant figure out my problem. Also the problem being that I need a high receptor expression level as a consequence of transfection as I do binding studies.

Thanks for you reply,,
Tc

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Try making 2x HBS (0.5 L) with different pH (slight variations) and try transfections to identify which pH gives best transfection and stick to it. One has to optimize calcium phosphate transfections and it needs a bit of time but is cost effective in the long run.

good luck !!!

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60-70% is a very good transfection efficiency. You dont get much better even with lipo2000. Problem may be due to other reasons. I know CaP04 method has a lot of varibility ( lipo2000 is too, but less than CaPO4). Check if you can incorporate a reference plasmid to tell you the true transfection efficiency each time. You need some stuff DNA anyway. Just add some EGFP construct there you know if it is a transfection problem or other reason.

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Hiya,

Well, I dont use junk DNa and just use 20ug of DNA i want. Also, i like ur idea but since I am focussed on receptor expression level, which is independant of transfection efficiency. I also fear GFP expression may use transcription factors and stuff resulting in hijacking of the expression of my desired transfected protein.. Am I making any sense.. am not quite sure..

Thanks!

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