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Creating a stable cell line by transfecting a plasmid without any fluorescence t


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#1 science noob

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Posted 20 August 2012 - 12:19 AM

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?

#2 klenow

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Posted 20 August 2012 - 02:06 AM

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,

Edited by klenow, 20 August 2012 - 02:06 AM.


#3 science noob

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Posted 19 September 2012 - 06:46 PM

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.

#4 bob1

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Posted 20 September 2012 - 01:18 AM

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.




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