Want to know how transfection of mutants in cell lines can be performed in effective manner? Any detailed protocol which would be cheap and easy to perform kindly suggest.
Robert Adolf Brinzer
Using calcium phosphate nanoparticles can be a cheap way if electroporation is not an option.
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Anatoliy Zubritskiy
Please keep an eye on the pH of Ca buffers, the 0.1 pH shift may drastically change the efficiency.
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Bandla Ramesh
Mutant plasmid DNA containing the desired gene or genetic mutation. Tissue culture-treated plates or dishes appropriate for your cell line. Calcium chloride (CaCl2) solution (2.5 M). HEPES-buffered saline (HBS) solution (pH 7.05). 2x HEPES-buffered saline (2x HBS) solution (pH 7.05). Complete growth medium appropriate for your cell line. Sterile water. Prepare Cells:Culture your target cell line in complete growth medium until it reaches approximately 70-80% confluency on the day of transfection. Prepare DNA-CaCl2 Mixture:In a sterile microcentrifuge tube, combine 1-10 μg of mutant plasmid DNA with 250 μL of 2.5 M CaCl2. Adjust the volume with sterile water if necessary. Mix the DNA and CaCl2 solution by gently vortexing. Prepare HBS Solution:In a separate tube, prepare the HEPES-buffered saline (HBS) solution by diluting 500 mM HEPES to a final concentration of 50 mM with sterile water and adjusting the pH to 7.05. Add DNA-CaCl2 Mixture to HBS:While vortexing the HBS solution, add the DNA-CaCl2 mixture dropwise to the HBS solution. Incubate the mixture at room temperature for 15-30 minutes. This allows the DNA-CaCl2 precipitate to form. Transfect the Cells:Remove the growth medium from the cultured cells and add the DNA-CaCl2-HBS mixture dropwise to the cells. Gently swirl the plate to ensure even distribution of the transfection mixture. Incubation:Incubate the transfected cells at 37°C in a 5% CO2 incubator for 4-6 hours. Remove Transfection Medium:After the transfection period, remove the transfection medium containing the DNA-CaCl2 precipitate and replace it with fresh complete growth medium. Allow Expression:Incubate the transfected cells for the desired time to allow mutant gene expression or protein production. Analyze Transfection Efficiency:Analyze the transfection efficiency using appropriate methods (e.g., GFP expression if using a GFP-tagged mutant). Subculture and Maintenance:
Hi, try this protocol
Materials Needed:
Protocol:
- Continue to culture the transfected cells using standard cell culture procedures for further experiments or analysis.
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