Transfection - Something wrong with transfection? (Nov/12/2007 )
I have transfected a cell line with MUC-1 cDNA. and I am sure the cells are stably transfected by FACS analysis. However, when I lysed the cells and do PCR, I could not see the right band of the DNA on PCR. Does this PCR actually tell me that there is something wrong with the transfection ?
Tell us how did u establish the "stable" cell line step by step.
Thank you for help. This is how I developed stable cell lines. The pcDNA/MUC1 was transfected into C57MG via Lipofatamine transfection reagent. Cells were plated at 70-80% confluence on day one. On day tw, plasmid DNAs were mixed with Lipofactamine, and added dropwise to the cells in serum-free Opti-MEM medium. About 8h after addition of DNA-Lipofactamine, the cells were fed with equal volume of growth medium with 2x serum. 48 h after transfection, G418 was added to final concentration fo 1.5 mg/ml, and cells were grown for 7 days in G418 selection medium. Single clone selection was perfomed by limited dilution with 96-well plate in normal medium supplemented with 0.75 mg/ml G418 for 2 weeks. G418 resistant single clones over expressed MUC-1 was selected based on confirmation by Western blot and FACS and then expanded.
Is this true for one clone or all clonies? Some recombination events may occur during DNA integration. Can you use a different set of primers for MUC?
Probably you are right, because the primers I am using is for a whole MUC-1 genome, and MUC-1 cDNA from transfection may require different primer