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unstable retroviral plasmids

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#1 indu



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Posted 23 March 2010 - 09:52 PM

Hi Guys, i'am frustrated with this unexplainable results i get with my retroviral constructs (pMIG backbone, that has an IRES driven GFP as internal reporter)....every time i make a fresh midi prep and i check for expression in HEKs by lipofection, i face this problem....the first transfection works and i get good GFP expression 60-70%.....when i freeze the plasmid and reuse it for subsequent transfections, the efficiencies go down drastically to 5-10% or even less.....i really don't know whether the plasmid is becoming unstable by repeat freeze thaw (i store at -20)....the host strain is DH5a....i do the culture at 30C to avoid any recombination and include Amp 50 selection right from the transformation stage.....the plasmids were also checked by restriction and seems to be fine...though the yield varies each time between 100-300ug from a 100ml culture....the total plasmid size is 7-8kb and the insert sizes between 1-2kb...can they become unstable after isolation and during storage?....can anyone explain this strange thing?

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