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Puromycin selection versus FACS


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#1 seanspotatobusiness

seanspotatobusiness

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Posted 24 March 2014 - 05:11 AM

I want to select for my transfected cells. My plasmid encodes puromycin resistance and a fluorescent protein. Any thoughts on what route to take to achieve a fairly pure population of transfected cells?



#2 bob1

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Posted 24 March 2014 - 11:32 AM

You need the selection pressure to ensure that your cells retain the plasmid, so adding puro is essential.  From there you can go either of two routes - select and do a clonal expansion (make a monoclonal line with your insert), this is slow but the classic way of doing it and makes characterization easier.   The other route is to sort by FACS the cells that glow with the fluorescent protein and have maintain the selection pressure to produce a polyclonal line with multiple cell populations mixed into one.  This is faster but mixed populations typically have variable insert expression levels.






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