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        Transfection of siRNA oligos to peritoneal macrophages

        互联网

        979

         

        Use DeliverX Transfection from Panomics

        Modified protocol below

        Step 1:  Make complexes

        Note:  Do not scale up more than 3x

        Thaw siRNA and DeliverX reagent

        Dilute siRNA into buffer 1

        10 uM siRNA  2.17 ul

        Buffer-1    47.8 ul

        Sonicate DeliverX Reagent maximum output for 3-5 min

        Dilute into buffer 2

        DeliverX Reagent  16 ul

        Buffer 2  34ul

        Sonicate again for 3-5 min

        Mix the diluted siRNA and the diluted DeliverX

        Vortex

        Incubate at 37 deg C for 20 min

        Use immediately or store at -20 deg C for up to 6 months (<10 freeze/thaw)

        Mix equal volumes buffer 1+ buffer 2

        Add 300 ul mixed buffers to concentrated complexes (total 400 ul)

        300

        Step 2:  Transfect cells

         

           96 well  24 well  12 well  6 well
         Pellet Cells, wash PBS, Pellet Cells
         Resuspend Cells @ 3333 k/ml in serum free media
         Add siRNA working solution to plate  30  150  200  300
         Add cells in serum free media  30  150  200  300
         Incubate 4 hours        
         Add complete growth media  100  500  670  1000

         

        Incubate 48-72 hours

         

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