Calcium Phosphate Transfection - (Apr/19/2006 )

Hi all,

Just a small question really. I have been using HEK293 cells for a few years. When using calcium phosphate transfection I tend to get a transfection efficiency of 30-70% (depending on plasmid size). I am happy with my protocol however I was wondering if anyone has any tips on how to increase this yield further?

I transfect cells at about 80% confluency in a 6 well plate. Combining the plasmid DNA (high purity) and water first before adding calcium chloride. I add this to 2xHBS while vortexing, adding the DNA-CaCl2 a drop at a time. I leave at room temp for 25mins, vortex again, then add to my cells. All reagents at the start (apart from DNA) are warmed to 37degrees.

Just wondering if anyone knows of any tricks that seem to increase transfection efficiency further? I am not keen on using Lipofectamine.

Many thanks,

Deb

Dear Deb:

hi, my name is valeria and i'm very suprised with the efficency of yours calcium phosphate transfection, i'm working with primary cultures and they had not more than 4 - 8 transfected cells bye plate. For these reason i want to ask you, why did you said that the size of the vector that are you transfecting is important? what's the size of your vector? because i have been trouble to transfect a vector that design in the lab who has 14 kb. I will really be so glad if you can answer muy question because i have been working in it for a month and it's a long time

and Best regards


Valeria

*for debbifry : you have a high percentage of transfection efficiency, which i don't suppose would be that enhaced. Congratulations for your efficiency (and i would be very happy if you could send me your protocol )


*for valerie : primary cells are very hard to transfect at more than 10% Best is to switch for an infection process with retroviral systems.

hi deb

In the viral core here, they use calcium phospghate to transfect 293T and get close to 90% transfection. When I tried their protocol, if lucky, would get between 30-50%, many times less than 10%.

May be u could send me ur protocol. Thanx.

A protocol I use for calcium phosphate with very high succesrates (using a 15 kb plasmid)...

On the day before transfection seed 700.000 cells in a 6 cm dish (5 ml high glucose DMEM with 10% FCS, buffered with bicarbonate, leave @ 37°C in a 4,5* CO2 incubator)

Then I use Endotoxin free plasmid DNA (Qiagen maxiprep), 12 µg per transfection (a lot, I kow). And I dilute my DNA into a total volume 219 µl H2O. Then I slowly add 31 µl 2M CaCl2, gently swirling around with the pipet tip to make sure the CaCl2 is distributed evenly in the solution (notice the higher viscosity of the CaCl2 versus the water). Then I add the total of 250 µl to 250 µl of 2xHBS (pH=exactly 7) in a polystyreen tube (stratagene claims polystyreen gives better results than polypropylene or polyethylene, don't know the reason and haven't compared myself) in a dropwise manner (again rather slowly). I mix this solution by blowing air bubbles with the pipet (3 times 1 ml of air bubbles). I leave this at room temperature for 20 minutes and then add this to the cells gently swirling around to make sure the precipitates cover the entire surface. Incubate overnight and the next morning I replace the medium with fresh complete D-MEM.

Haven't FACS analysed transfectin efficiency, but with fluorescence microscopy I notice that efficiency is very high.