I am culturing MCF7 cell line and want to transfect the plasmid (GFP Tagged). Seniors says it is to be optimized. What is Optimation of transfection and how one can get higher transfection efficiency?
It depends on the transfection reagent that you use. You need to adapt the ratio DNA/transfection reagent to your cells and test different conditions.
transfection efficiency depends upon varius factors i.e. cell density, transfection reagent and DNA concentrations, transfection reagent-DNA complexing time, and media components (serum and antibiotics) .Also you need to screen the cells at various post transfection time points for getting maximum expression of the protein.
this link will give you some idea how to proceed with the experiments .........Good luck.....
https://ja.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.2473.File.dat/Focus%20Volume%2021%20Issue%203.pdf
use LDL. We tested LDL as a transfection vehicle using several different human cell types and a GFP-plasmid. in our experiments we tested for delivery first, i.e. does the plasmid get into the cell. to do this we labeled the plasmid with BOBO1 and followed time course delivery by fluorescence. this assured us that the plasmid was in the cell nucleus. basically we considered LDL to be similar to a CD or flashdrive which delivers the software and has no other agenda. next, we establish conditions and time required for the MCF7s to express GFP. with some cell types all of this is rapid. good luck.
LDL is available from several commercial sources but it is easy to isolate. send email to my [email protected] if you have questions.
Try this drug called enoxacin. There is a RNAi Nature paper..where they show incubating the cells 48 hr prior to transfection increases the efficiency.
We just starve cells for 2-4 hrs or we incubate overnite in medium with statins.
Hi,
I work with MCF-7 cells too and use Viromer RED for plasmid transfection (from Lipocalyx). I got very good efficiency.
@Alex
Lipofection is surely less toxic than viral transduction or electroporation but it may induce some negative effects to the cells too.
Many people report inconsistency of their transfection and some changes on cells, even if cell viability is not affected. As lipid-based, interactions with the cell physiology can be oberserved resulting in some troubles downstream the transfection.
Among chemical reagents, there is an alternative which is using polymer-based nanoparticles (e.g. PEI-derived reagents). Efficiency is equal or superior to lipofection, the workflow is the same (dilution + mix with your payload + incubation 24-48h) but clearly, toxic effects are not so important (transfection stays of course a perturbating event for the cells).
And do not forget the price. Lipo*******ne and others are really expensive!
To give some input to the initial question of Prem, I recommend reading our instructions' manual. From page 9, you can find a friendly optimization guide, which is applicable not only to our Viromers but to all chemical reagents.
Best,
Sandra
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