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Transfection of siRNA oligos to peritoneal macrophages
關鍵詞： transfection sirna oligos來源： 互聯網
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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
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| ? | ?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 |
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Incubate 48-72 hours
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