Hello everyone, I am currently doing transient transfection. I want to transfect the overexpressed plasmid into HCC cells, and this plasmid has 10000+ base pairs. The transfection reagent I used was lipo3000. After transfection, the empty vector group had green fluorescence, but the overexpression group had no green fluorescence, and the plasmid was increased from 2.5 μg to 5 μg, but the result was still ineffective. I wonder what aspects I should consider?
The specific operation is as follows:
the first day of six-hole plate laying plate;
Two hours before transfection, double antibody-free serum-free medium was replaced. Tube A (opti-mem 125 microliter, plasmid 5 microliter, p3000 10 microliter) was left for 5min, tube B (opti-mem 125 microliter, lipo3000 10 microliter) was left for 5min. Tube A was added to tube B and left for 20min. Pour the mixture drop by drop into the 6-well plate;
After 6 hours, the medium was replaced with complete medium.
Thank you in advance!
What's your transfection efficiency in the empty vector? In my experience, you can still transfect cells that are mycoplasma position albeit at a much lower efficiency. Have you checked your cells for mycoplasma contamination, it is a known source of inhibition and more common than people think. I've experienced it myself.
Hi, Can Kiessling
The transfection efficiency of the empty vector group was very low. My cells seemed to be infected with mycoplasma, but I didn't test for it. Can I use mycoplasma inhibitors to improve the efficiency of the experiment? How did you solve it?
Thank you! ₍ᐢ..ᐢ₎♡
I would highly recommend testing it, if your current cells have it then it is possible also the stocks have it, which in turn imply whoever sent the stocks might also have it. That is what happened to me, we found out all our stocks were contaminated even for different unrelated cell lines which came from another institution. We used to following kit for detection though you can find other kits that will do the same: https://bioscience.lonza.com/lonza_bs/TR/en/Cell-analysis/p/000000000000186472/MycoAlert-Mycoplasma-Detection-Kit-%28100-Tests%29
Then we treated the cells to get rid of the mycoplasma, we even tested the stocks in liquid nitrogen and found out that it didn't start in our lab, we just bought it over from the other institution where the stocks came from! It took much effort but we overcame it. Also if your tests become positive, consider throwing everything out and starting over instead of mycoplasma elimination. We really spent a lot of effort with testing & treatment to re-establish our stocks. But in the end, we boosted our transfection efficency from 5-10% to 40-60%. There are many kits for mycoplasma elimination, here's one from Lonza if you decide to stick to their detection kit also:
https://bioscience.lonza.com/lonza_bs/CH/en/Cell-analysis/p/000000000000186484/MycoZap%E2%84%A2-Mycoplasma-Elimination-Reagent-%285-Treatments%29
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