i need some help setting up my culture plate having negative controls,I am going to infect HEK293T cells in a 6-well plate... to one well I will add my lentivirus particles(packaged already), and to 2nd well, I will not anything and let only HEK293 T cells grow, and to the 3rd well can I add the empty lentivirus vector(having GFP and PUROMYCIN gene)? if yes,so will I add it without the packaging and envelop plasmids? and how this 3rd well can act as a control.
Hey,
I don't think that the empty lentiviral vector will work by itself. Even if it is an empty vector, one needs to add packaging and envelope plasmids. First you can make the LV particles having empty vector, collect and quantify them, and then use these viral particles as control in your cells
Hi,
The best control for your experiment in this case is to add all the plasmids, only changing the the plasmid with or without the insert. You want to keep you controls as close as possible to your experimental sample, only changing one variable. The 2nd control (untreated HEK293T cells) would only be relevant if you suspect that the transfection is somehow affecting your readout, which could be the case. You can also just take it along for peace of mind.
In summary, I think the best control would be well 3 with all the plasmids.
Good luck with your experiment.
Thank you very much for your response,but the thing is if i use an empty lentiviral vector along with the envelope and packaging plasmid,so will this well give me egfp fluorescence?? if yes then how i can compare this fluorescence with that of the well in which i added lentivirus wij ky gene of interest along with all envelope and packaging plasmids? because both of the vectors contain egfp reporter... Emma CL Cook Sreelakshmi S Kumar
Hi, it depends on your readout. Is GFP fluorescence your readout? Then yes, this would be an issue and the ideal thing would be to have a vector with no gfp. However, notmally the gfp is not a problem. If you are looking at the expression of mRNA of a certain gene, for example, you can use gfp to check your transfection efficency. Transfected cells will become green, and you want similar transfection efficiency between your control and your experimental sample.
If you really want to avoid gfp, and you don't have any other plasmid to substitute it, then I would recommend transfecting only the other 2 plasmids. At least this way the control cells will go through the transfection process.
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