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?

Hi,
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

sweetdaisy1975

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