Creating a stable cell line by transfecting a plasmid without any fluorescence t - (Aug/20/2012 )
I have a plasmid which I need to transfect stably into HEK cells and the plasmid does not contain GFP/YFP etc or any other luminescence promoters. How would I go about selecting cells containing my plasmid of interest?
The plasmid should contain some gen that confers resitance agains an antibiotics (puromycin resistant). If this is not the case, you should clone your cDNA into a more appropiate vector because although you can do stable cell line with your plasmid, you will have to check for expression everytime you want to use the cells (and without a visual marker that means to perform PCR or better Q-PCR if you want to compare levels and/or Western blot)... In my opinion, instead of doing all that stuff before any experiment, I would prefer spend one week and clone the cDNA in another vector...
Best,
klenow on Mon Aug 20 10:06:06 2012 said:
The plasmid should contain some gen that confers resitance agains an antibiotics (puromycin resistant). If this is not the case, you should clone your cDNA into a more appropiate vector because although you can do stable cell line with your plasmid, you will have to check for expression everytime you want to use the cells (and without a visual marker that means to perform PCR or better Q-PCR if you want to compare levels and/or Western blot)... In my opinion, instead of doing all that stuff before any experiment, I would prefer spend one week and clone the cDNA in another vector...
Best,
The plasmid does contain a neo resistance. And for your info, I did not construct this plasmid and we got it from another lab - this is a commonly published plasmid.
Just use neo selection and assay for protein of interest production - western blot of immuno of some sort. You'll need to select individual clones and grow them up before assaying, which will take some time. You could also create a pooled clone, where you select a bunch of colonies, pool them and then assay - its a bit quicker but has the risk that expression might be lower.