i have a question about electroporation with gene pulser xcell, i electroporate huh7 cells , with170 v,40ms 4mm cuvette, dmem (without fbs and antibiotics) as electroporation buffer, but i get very low transfection efficiency.
my question is , should i use pre-warmed complete medium( dmem+ 15% fbs) without antibiotics(pen/strep1%)? does antibiotic have effect on transfection efficiency?
thank you
This is one year later, I hope you were able to solve the electroporation issue. Nevertheless, it is best to use Optimem medium for electroporation. First, wash your cells in Optimem, pellet and resuspend in the requisite amount of Optimem then add your DNA and incubate on ice for 10 min, shaking the cuvette from time to time to ensure even distribution of cells and DNA. Electroporate with the above conditions and incubate again on ice for 10 min post electroporation. Resuspend in warm complete medium, without antibiotics and culture at 37'C. Antibiotics are known to be cytotoxic, especially in transfected cells (electro- or lipo-transfection). Hope this helps.
Hesbon Amenya Zablon@ Hesbon Amenya Zablon thank you so much.
i solved the efficiency problem but still i have another issue, which is contamination of cells after a month after G418 selection, when i transfect cells as stable.
do you have any suggestion ? i read that maybe ethanol precipitation can sterilize dna . but i am concern about the salt which is added to dna and its effect on transfection.
i would be appreciated if you could suggest any experience.
Hello Gilar gorji bahri I am happy you were able to take care of that. Are you dealing with bacterial contamination of your culture? It would be highly unlikely that it is arising from the plasmid DNA, as all bacteria are lysed during plasmid preparation. I would suggest you consider the aseptic procedures during culture, and decontaminate your hood and incubator.
It is likely that there is some salt carry-over during ethanol precipitation of plasmid DNA, but in our hands, I have not seen a decrease in transfection efficiency even after ethanol precipitation. Another option you have is to precipitate the plasmid DNA with isopropanol (http://cshprotocols.cshlp.org/content/2017/8/pdb.prot093385.full). If you are using a commercial kit (e.g. QIagen mini, midi, maxi)for your plasmid DNA preparation, then there is no likelihood of having bacterial or salt contamination of your DNA. All the best!
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