Dear all, I'm trying to transfect HeLa cells with lipofectamine 3000 in a 10 cm dish. Do someone of you have a protocol to share? I still examined the table for scaling up or down lipo3000 but it gives wide concentrations range:
DNA14–28 ug
P3000 28–56 ul
lipo 3000 21.7, 43.4 ul
Thanks in advance
Hello Francesca Bertino ,
If it's the first time transfecting your plasmid I would highly recommand to do an optimization in a smaller plate, like a 24-well plate. In this plate, you'll try several concentration of your plasmid with different ratio of Lipofectamine:P3000 while also adjusting ratio between those reagent and DNA quantity.
As a control of your transfection you can use an empty-GFP plasmid.
If you have already tested the transfection with your plasmid in smaller plate within HeLa cells, you just have to scale up the quantity of DNA based on what you are using in smaller plates.
As an example, I use 0.25 µg of plasmid per well in a 24-well plate and I scale it up to 9 µg in a 10 cm dish. Then, I keep the same ratio between my DNA quantity and Lipo/P3000 reagent that was optimized before, which is in my case 1.5.
It brings me to use 9 µg of DNA, 13.5 µL P3000 and 13.5 µL Lipofectamine for a 10 cm dish.
If your plasmid is precious, you can even decrease the DNA quantity.
Hope it helps.
Maxime.
I did on 293T cells in a 10cm dish,
5ug of DNA, 10ul of P3000, and 5ul of lipofectamine.
works great for co-IP.
I think you might try
10ug of DNA, 20ul of P3000, and 10ul of lipo. since 293T is too easy.
hi
I agree with maxime , it seems lipo 3000 very effective for HeLa transfection .
Note : you should used from maxiprep plasmid to getting high rate of transfection (concentrate DNA minimum 1ug/ul and high quality )
this formula maybe ok :
DNA15 ug
P3000 40 ul
lipo 3000 32 ul
http://www.accesolab.com/wpress/wp-content/uploads/2014/07/manual-lipofectamina.pdf
good luck
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