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Calcium transfection problems - (Dec/06/2006 )

Hi all,

I have some problems with my calcium transfection of HEK 293T cells.
My protocol is: (1 well of 6-well plate)
3,5 ul DNA
44,5 ul 0.1xTE
32,0 ul MQ
9,0 CaCl
----- 89ul

Vortex and add by continous vortexing 89 ul HBS

Pipet at cells.

The pH of my HBS buffer is arround 7.12

After the transfection I see a lot of small particles, think some kind of membrane structures in my medium, and also a lot of cells are dying very fast and de-attach from my well. The efficiency of my transfection is very low, which makes it inpossible to do futher experiments...

Does anybody know what could be wrong with my protocol?

-Roy van Heesbeen-

the particles you see are okay, these are the calciumprecipiates. I also see them after the transfection of my cells. It seems that your cells do not like the transfection and therefore they might die. I use the following protocol:
- seed cells the day before so that the cells are 50 - 60 % confluent on the next day
- 2 h before make a media change
- transfection for 1x 6well:
1.5 - 3.0 ug DNA (purified by e. g. Qiagen colum)
12.5 ul 2.5 M Calciumchlorid solution
add to 125 ul 0.1 x TE buffer
add 125 ul 2 x HBS (281 mM NaCl, 100 mM HEPES, 1.5 mM Na2HPO4, pH 7.12 (store at -20C)
1. mix TE buffer and DNA
2. add Calciumsolution and mix
3. add dropwise!!! 2 x HBS vortex!!!!
4. add 250 ul of mix dropwise to cells
5. incubate 37C for 16 - 18 h
6. wash 1 x PBS and do a media change
7. use 24 - 72 h after transfection for experiments



make dif. pH HBS and try them out side by side and select the one with the best efficiency.


I have also the problem with calcium phosphate transfection. I used COS7 and HepG2 cells, after transfected, all cells are lysed and died. I try it for a long time, but still fail.
I use 37 ul 2 M CaCl2
263 d.H20 (include DNA)
300 ul 2x HBS
and add the 200 ul precipitate mixture to 60 mm. plate culture

How can I do then? Please Help me. Thanks