I am trying to transfect RL34 cell lines using CaCl2 Transfection method. But, seems cells are finding it hard to uptake the DNA. Any Idea about Transfection factor optimization for RL34?
Try this (optimized for 60 mm plates, in my experience it yields very high transfection efficiency in HEK-293 cells):
1) cells should be approximately 80% confluent prior to transfection
2) just prior to transfection, change the medium to 4 mL of complete Growth Medium (GM)
3) in an eppendorf tube, prepare the transfection cocktail by adding 10 µg DNA to dH2O (the final volume must be 438 µL). Add 62 µL 2M CaCl2 to the DNA/H20. Add 500 µL 2X HBS (pH 7.05) by bubbling. Within 1-2 minutes add this solution to the cells and gently agitate to insure uniform mixing. Return the cells to the incubator.
4) Optional: approximately 10 hours after adding the DNA, remove the medium and gently replace with fresh GM
5) Analyze the cells 24, 48 and 72 hours post-transfection
Reagents
- HBS (2X): 50 mM HEPES, pH 7.05; 10 mM KCl; 12 mM Dextrose; 280 mM NaCl; 1.5 mM Na2HPO4 (FW 141.96). The final pH of the solution should be 7.05 +/- 0.05. Filter through a 0.2 µm filter, aliquot, and store at -20°C. Avoid multiple freeze/thaw cycles.
- 2 M CaCl2: prepare a 2M solution and filter through a 0.2 µM filter, aliquot, and store at -20°C.
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