Neuronal Primary Cell Co-Transfection via Calcium Precipitation

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Neuronal Primary Cell Co-Transfection via Calcium Precipitation

I must be doing something wrong with my Ca+ Precip. transfection because even my control has no usable surviving cells.  This is the methods I am following:

Primary hippocampal neurons were transiently transfected by using a modi?ed Ca+2 -phosphate precipitation method. The media of cultures varying from 7 to 11 d in vitro (DIV) was changed to DMEM containing 1 mM sodium kynurenate and 10 mM MgC l2 (DM EM / Ky-Mg 2 ) and was incubated for 30 min at 37°C /5% C O2. During this incubation Ca 2 - phosphate/ DNA solution was prepared, 60 ul containing 4 g of DNA (3 g of the RNA vector; 1 g of GFP vector if two plasmids were transfected); 250 mM CaCl/DNA was added drop-wise to 60 ul of 2x HBS [containing (in mM) 274 NaC l, 10 KC l, 1.4 Na2HPO4, 5 D-glucose, and 42 HEPES, pH 7.07]. Precipitate was allowed to form for 20 min, and 20 ul was added drop-wise to 12 mm coverslips. Cells were incubated at 37°C /5% C O2 for from 30 to 90 min, washed once with DMEM / Ky-Mg 2 , once with Neurobasal contain- ing B27 and glutamine, and returned to conditioned Neurobasal medium. Coverslips were used for microscopy in 24 – 48 hr.