I am trying to transfect Hela cells with a large 12 kb plasmid by using Lipofectamine 2000. I have used various concentrations (1,2,3,4 and 5 ug) of the plasmid and transfection reagents but the maximum efficiency was 30 %. I am not sure if I can get higher efficiency with such a large palsmid?
Can someone answer please whether this efficiency is sufficient to check the expression of genes transcribed by the transfected plasmid? Also, help me knowing which reagents work best for the Hela cells?
Hello Naveed Shahzad
You could improve on the efficiency.
Make sure you have included the points below:
1. Transfect cells at 40–80% confluency. Too few cells may cause the culture to grow poorly without cell-to-cell contact and too many cells will result in contact inhibition, making cells resistant to the uptake of foreign DNA.
2. Be sure that the passage number is below 40-45.
3. Be sure the DNA is free of protein, RNA, chemical and microbial contamination as impurities could decrease the efficiency.
I would recommend you to use jetPEI which is a powerful reagent that ensures robust, effective, and reproducible DNA transfection into mammalian cells with low toxicity.
Please refer to the link below for more details.
https://fnkprddata.blob.core.windows.net/domestic/data/datasheet/PPU/101-10N.pdf
Good Luck.
Malcolm Nobre Thank you for your answer. I have already considered all of these factors while transfecting my cells. However, I have not tried the Polyplus Jetprime transfection reagent. I have also seen in multiple publications that it works well with HeLa cells. So, I think it would be a good idea to try it.
Thank you
I agree to Malcoms comments.
However, in my opinion Lipofectamin(R)2000 ist the most adequate transfection solution. Using this method, you have to modulate different stepts (as mentioned by malcom) and have to check the outcome/result of each variation.
Higher efficacy could be reached via using other methods (e.g. electroporation with AMAXA apparatures should lead to transfection efficacy of 80-95%, but are also associated with an increased loss of cells).
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