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circular or linear pDNA for stable clone generation?


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#1 mercury

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Posted 05 April 2010 - 08:09 AM

Hello all,

I had argument with some of my lab collogues about generating a stable clone using circular or linearised vector. Most of them insisted that circular electroporation will lead to transient tansfection, although in my experience, I have generated close to 25 different cell lines using circular vector (w/o linearising it with RE). What’s your opinion?
Appreciate your efforts to write back

Thanks so much
m

#2 pDNA

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Posted 05 April 2010 - 09:22 AM

its both possible ...but to my knowledge (i'm not that big specialist in cell culture) linearized DNA gives you more "control" over the integration event ...the plasmid will be linearized by cell intrinsic endonucleases anyway ...if you use circular pDNA there is the increased chance that the plasmid gets linearized in a region that is detrimental for your experiment ...so it is possible that you generate clones that do express the marker gene but not your gene of interest (because the plasmid was linearized in this region).

Generally, linearized pDNA has a lower transfection efficiency than supercoiled pDNA because of its size.

Regards,
p




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