i check the condition of the cells. But I get the worst condition the cells. Only a few cells that attach on TCD.
so, may you help me to solve electroporation problem?
thanks
Dear Dyaningtyas: I think you are adding too much of DNA, please titerate the DNA first, starting from 0.1 micro-gram
Final concentration is 8mg/ml but what about the actual input amount? And how many cells. I believe most electroporation protocols using the standard cuvettes require roughly 1x10^6 cells and total of 10microgram of DNA. That is a good starting point. Did you optimise the parameters for electroporation?
It is important to immediately mix the cells with serum containing medium after electroporation.
dear amita ajit, thank you for your suggestion. Yes I do like your suggestion. Is it better transfection using chemical compare using electroporation?
dear callum parr, I'm sorry to typing microgram. I'm using plasmid with final conc 8 microgram/ml. My previously friend had already optimize it. Anyway what is critical step when transfection using electroporation? For the others cells, I optimized voltage, ms and pulse. Higher voltage, ms, pulse cause death cells.
dear evelyne nguyen. Yes I've optimize it.
sorry for asking here should i use pre-warmed complete medium( dmem+ 15% fbs) without antibiotics(pen/strep1%)? does antibiotic have effect on transfection efficiency?
Yes, antibiotic has effect on transfection efficiency. You should use complete medium without antibiotic.
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