After standardizing the optimized puromycin concentration (concentration at which nearly all cells are dead between 5-7 day), 48 hrs post transfection antibiotic was added to the cells at three different concentration (lower, optimal and slightly more than optimal; just in order to increase the chance of getting good selection in either of these 3). But it happens that after 5 days of selection only 5-6 cells are GFP positive, though the transfection efficiency was not that bad (around 35%). What is wrong with the protocol?
Transfection efficiency gives you the rate of cells which transiently express your GFP marker. Upon selection with Puromycin, you are actually selecting those which have stably incorporated your plasmid in their genome. Stable integration rate is much lower, so there is not a direct correlation with your transfection efficiency. If you want to increase number of stable cell lines you get, you may try to linearize your plasmid from a safe place, it will increase the chances for the plasmid to be integrated without disrupting its constituents.
Transfection efficiency gives you the rate of cells which transiently express your GFP marker. Upon selection with Puromycin, you are actually selecting those which have stably incorporated your plasmid in their genome. Stable integration rate is much lower, so there is not a direct correlation with your transfection efficiency. If you want to increase number of stable cell lines you get, you may try to linearize your plasmid from a safe place, it will increase the chances for the plasmid to be integrated without disrupting its constituents.
thank you Hakan Bagci for you answer. Could you suggest me one more thing that in some protocol people use directly the restriction digested DNA for transfection while some use purified DNA. Do you have experience with it, which one will be better?And also about the transfection reagent as in this case i am using amaxa nucleofector, will with linearized plasmid lipofectamine a good option as in nucleofection we need to keep the volume upto 100µl -110µl (including their reagent and pur plasmid).
Hi Sangeeta, you should ideally run your linearized DNA on an agarose gel and extract by gel extraction (several kits are available), and then do an ethanol precipitation to dissolve the DNA in small volume of water or TE buffer (At this step, you may want to run again the linearized DNA on the gel, to see whether it underwent religation, although it is a low possibility in case this occurs you may want to add a dephosphorylation step after restriction digestion). This will give you a relatively high concentration to be used with Amaxa Nucleofector. You can alternatively use lipofectamine, but in my experience electroporation works better as it targets your DNA straight to the nucleus.
I agree with Hakan, the percentage of cells that stably integrate the plasmid is very low (usually on the order of 0.1% of transfected cells), so once the plasmid is lost from the transient expressors, the cells will start to die. When we are doing this in our lab, we use circular plasmid (linear may work better, but we haven't tried comparing them), and we transfect 4 different 10 cm dishes. After 2-3 weeks of selection, we then pick colonies and transfer them to a 24 well plate for further expansion and screening. This usually gives us a large number of stable clones to choose from.
Good luck!
Thank you Hakan and Jonathan for your kind inputs. I will try with linearized plasmid, let s see how it goes.
Hi Hakan, indeed linearized plasmid DNA is more efficient for stable integration in the genome than circular plasmid. However, doesn't the uptake of the DNA by the cells drastically decrease when using linearized plasmid ?
- Badges
- Science method