I need to cotransfect two plasmids
1. GFP conjugated with protein of interest and
2.YFP conjugated with protein of interest.
Control
5ug of GFP plasmid transfection in 1 well
5ug of YFP plasmid transfection in another well
My question when co-transfect do the plasmid needs to be equal (final 5ug all the case ) or double (10ug in case of cotransfect) as the pore formation for plasmid entry by lipofectamine 3000 is going to be same which one is right
Do i need to take 2.5ug of my GFP plasmid DNA and 2.5ug of YFP plasmid and use it with 5ul of lipofectamine 3000 final : 5ug
Or
Do i need to take 5ug of my GFP plasmid DNA and 5ug of YFP plasmid and use it with 5ul of lipofectamine 3000 final: 10ug
which of the above is right and what is the scientific reason
whatever the lipofectamine protocol recommends- use half dose of one plasmid, and half- another. Keep in mind- its a good idea to optimize your transfection anyways, eg for a given total amount of plasmid- try 3 concentrations of lipofectamine, and see which condition maintains the best balance between efficiency of delivery, and cell viability. Luckily, both Lipofectamine 2000 and 3000 are "forgiving" and delivery efficiency is good in a broad range of conditions
Hi! Your transfection reagent will be optimized for taking up a certain amount of DNA. Thus usually you would split the total amount of DNA transfected and use half and equal amounts of your DNAs of choice (5 ug total = 2.5 ug DNA 1 + 2.5 ug DNA 2). This is what I did when using Lipofectamine LTx and 2000. However, these are starting conditions. You may find you have to increase the amount of one of your constructs? This depends on several factors, including the size of your constructs and how well the cells tolerate that particular protein. As suggested above, you have to optimize all concentrations depending on your constructs and the type of cells you want to transfect.
Hope this helps! :-)
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