I'm trying to transfect MCF-7 with 2ug caspase-3 GFP plasmid using Lipofectamine2000 and the transfection efficiency is terribly low. Can anyone suggest me something which can enhance the transfection efficiency ?
Dear Punita,
What is your cell confluence at time of transfection? Cells should not be confluent/ be at stationary phase at time of transfection. 70 to 90 % is fine for most adherent cells. The viability of the cells should also be greater than 90%. Do you use serum-free medium? Serum can interfere with complex formation and reduce the efficiency of your transfection. I used OptiMEM reduced serum medium from Gibco. MCF7 is not a "hard-to-transfect" cell line, however, you may want to try lower passage number cells. Have you thought of trying a reverse transfection protocol?.
This may also be of help:
https://www.thermofisher.com/za/en/home/references/gibco-cell-culture-basics/transfection-basics/factors-influencing-transfection-efficiency.html
Best
Phil
Dear Punita, there is the Lonza ready-to-use protocol available. Without any further optimization you get around 70% transfection efficiency with 89% living cells. This protocol and program can be used for DNA, siRNA, gRNA, peptide and protein transfection, Many people use this well established protocol for genome editing. Details can be found here: https://www.lonza.com/products-services/bio-research/transfection/nucleofector-kits-for-cell-lines/nucleofector-kits-for-cell-lines-k-m/nucleofector-kits-for-mcf7.aspx
Dear Punita, you can use this link;
www.lifetechnologies.com/transfection
Or MCF-7 transfection kit from Altogen Biosystems
Day 1: seed 90000-120000 cells per plate 100 mm2 to transfect
Day 2: To prepare separately:
175 uL optiMEM + 10 uL pDNA (10-30 picomoles)
15 uL optiMEM + 3 uL Lipofectamine 2000
wait 5-10 mins before mix @ RT
mix both by dropping with a 1000 uL gilson (NO VORTEX)
wait 20 min @ RT
In the meantime change medium with medium without antibiotics and FBS (1.5 mL)
after 20 min, add 200 uL of transfection mix by dropping with Gilson,
Change medium with complete medium after 4-6 hrs.
Check transfection efficieny after 24-48 hrs.
GOOD LUCK
http://www.thermofisher.com/es/en/home/references/protocols/cell-culture/transfection-protocol/transfecting-plasmid-dna-into-mcf-7-cells-using-lipofectamine-ltx-reagent.html
https://viromer-transfection.com/wp-content/uploads/MCF-7-transfection-viromer.pdf
Hi Punita, while this is common sense, I've found that being consistent in my cell culture (i.e. split ratio and passage interval) prior to plating makes a huge difference in transfection efficiency of MCF7 cells (more so than other cell lines I've worked with). Importantly, you want your cells to be actively dividing - I typically will plate when cells are 70-80% confluent, but not more than this. I also will avoid using cells above passage 12-15 post-thaw.
Another factor to consider, if you haven't checked already, is the possibility of mycoplasma contamination, which can make even the most easy to transfect cell lines (think 293T) near impossible.
Good luck!
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