You should be able to use the purified plasmid, if you have worked cleanly, especially using kits for purification I have never had problems with that. When introducing any component that has been opened outside of a sterile bench to my cells, I always used antibiotics on my cell culture to be very safe. Concentration wise, simply using a nanodrop (preferably) or spectrometer should help for the determination of the plasmid concentration, given that you know the length of your plasmid. Check for example here: https://nebiocalculator.neb.com/#!/dsdnaamt. There is no simpler way that comes to mind that will give the same accuracy. If you only need it very roughly, you could also run an agarose gel but I would not presume that to be simpler.

Hi Anja,

thank you so much for your reply! I did use Qiagen plasmid Maxi kit for purification of my plasmid. I just wanted to make sure i can use my plasmid using E.coli as my vector, purify it and then introduce them to my cells. I did use the corresponding antibiotics into my plasmid in my media agar

Hi,

You can use the amplified plasmid you extracted from E. coli to transfect your mammalian cells.

I am not working with mammalian cells myself (but some people of my team do, so I am slightly aware about these), but I don't think you can really "amplify" a plasmid in this type of organism. The advantage of using bacteria for the amplification step is their fast doubling time and the huge amount of cells you can reach in an overnight saturated culture (far higher than yeast, plant or mammalians cells). As each bacterium cell contain several copies of the plasmid, you can extract high amount of plasmid DNA from a small volume of culture.

After purification, DNA remains DNA, meaning that you can use it in any target organism. Of course, it depends on the selection marker on the plasmid. If you are working in mammalian cells, it should contain a gene specific for mammalian antibiotic.

The plasmid will not replicate in the mammalian cell. Certain plasmids can be selected for integration eg using the TRex system or a lentiviral/retroviral system followed by selecting for a marker for example gfp or puromycin/zeocin/ blasticidin etc. Otherwise the transfection is transient and the plasmid will be lost following replication.

Other issues to consider. Which primary cells are you working with? Best to look up in the literature how others in the field are transfecting. Standard transfection (lipid/chemical) based will fail in some primary cell lines, with high cytoxicity or very low transfection efficiency. Transduction or electroporation may be more successful/needed depending on your question.

Is your plasmid designed for expression in mammalian cells and if so does it have a selection marker?

Good luck with your experiment