hi
i want to make stable cell. i use A549. i tried to do transfection on plasmid. but it is difficult to make it. so i tried to make linear plasmid before transfection. but i am not sure i have to do extra purification after restriction enzyme cut. could you share your experience?
thanks.
Hi Oki,
In different cell lines transfection of non-linearized pasmid is usually sufficient to establish stable cell-lines. Linearization should increase the percentage of cells stably retaining your construct, as integration occurs more easily. However linearization and (purification, yes you should preferably purify your plasmid after digestion) is not going to solve the problem if the delivery is the major barrier (for instance if your cells are not easily transfectable). Instead you will add technical variables as poor DNA quality or quantity (which usually occurs after purification). Before trying again to linearize and transfect I would try nucleofection with your plasmid and prolonged selection, that should be sufficient to establish a stable cell line. But this is just a suggestion.
Good luck with your experiments
Hope it helps
Francesco
Hi Oki,
you will need your DNA to be of an acceptable quality for transfection. So, yes, definitely purify!
Good luck!
i tried to make transient state on that plasmid, which contains luciferase gene. but i tried to 1,3 and 5ug on 60mm culure dish for 1-2days . it doesn't affect on luciferin. neither circular plasmid nor linear plasmid worked. so i don't know what i have to fix it. could you give me some tips for it thanks.
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