Calcium phosphate transfection protocol

Protocol for calcium phosphate transfection of mammalian cells with siRNA.

This is derived from the protocol of Chen and Okayama, 1987.

Originally “told to us” by Harry Mellor (https://mellorlab.wordpress.com/). It works very well. As Chen and Okayama themselves point out, “the crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation”.

siRNA by calcium phosphate

Day 1

Trypsinize and count cells
Seed cells at 75000 cells/ml in DMEM with 10% FCS.

This does depend on cell type, growth rate etc.

Day 2

For a 10cm (10ml) plate prepare:
10 µl siRNA (20µM stock) in 500µl filter-sterilised 0.25 M CaCl2
Add 500µl filter-sterilised BBS (50 mM BES, pH 6.95, 280 mM NaCl, 1.5 mM Na2HPO4).
Mix by pipetting gently and leave at room temperature for 15-20 min
Add siRNA transfection mix dropwise to plate whilst swirling
Remove plate to a 3% CO2 incubator.

Day 3

Wash plate once with PBS then add fresh medium and return to 5% CO2 incubator

Day 4 or 5 depending on application

Harvest/do experiment

 

We usually leave cells for 72h for effective depletion but this is target dependent.

Works well with a variety of cells including HeLa and RPE1.

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