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Protocol for cell transfection using the calcium phosphate method.
Utilize the following mix components for all experiments. This protocol has been optimized for transfection of neonatal rat cardiac myocytes. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid. Add 40 μL per well immediately after plating (20 μl each of the luciferase plasmid with 20 μL of the beta gal mix). All mixing (except that which requires vortexing) should be done in the sterile cell culture hood.
Total volume | 400 μL | 600 μL | 800 μL |
|---|---|---|---|
Sterile 2.5 M CaCl2 | 25 μL | 37.5 μL | 50 μL |
Plasmid | 10 μg | 15 μg | 20 μL |
Sterile H20 | Total of 200 μL when added to above. | Bring to total of 300 μL with sterile H20. | Bring total to 400 μL with H20. |
Combine the above three reagents in a hood using a sterile 1.5 mL Eppendorf tube. Mix gently. Do not vortex. | |||
Sterile 2X HBS (hepes buffered saline) | Add 200 μL drop-wise with gentle agitation of the above mixture (setting of 2 on vortex mixer). | 300 μl added dropwise with gentle agitation. Then invert several times to mix completely. | 400 μL added dropwise. |
After adding the 2X HBS, cap the eppendorf and let stand at room temp for no less than 15 min. When ready to apply the mix to cells, combine equal amounts of the reporter plasmid with beta-gal control mix. Mix gently by inverting several times and add 40 μl of the mix to each well of a 24 well plate (this will require optimization depending on the specific experiment) swirling the cells gently to disperse the transfection mix into the media. For transfection of another plasmid/construct on 10 cm plates, a total volume of 300 μl of transfection mix into 6 ml of media works well.
Dr Richard Pattern
Tufts-New England Medical Centre
Molecular Cardiology Research Centre
Boston, MA