Transfection of siRNA oligos to peritoneal macrophages

 

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