Mutation screening - (Aug/08/2014 )
I was wondering if someone would willing to help me. At our lab, we obtained mice with point mutation of the gene of our interest, which is activated and stabilized by double strand breaks (DSB), but I do not see anything on Western blot. My project is to find out if in our mice is the mutation have some phenotype, if our gene is activated. I cultivated MEFs and irradiated by 5, 8, 10, 15, 20 Gy, but Western blot is without result. I cannot see difference between WT and Mutant irradiated MEFs.
Does someone have experience how to find out if the mutation occurs? Does someone immortalized MEFs? Do you know any technique how to distinguish the mutation (point mutation) in genome?
I just want to know if the point mutation in our mice have/will have some phenotype.
Thank you for your time. I really appreciate it.
More detail needed...
Is this a western that has worked previously (on mouse cells too?)? Did you have a positive control for the protein on your western?
Is the mutation Mendelian inherited? How do you know that the MEFs you cultivated were positive (+/+ or +/- not -/-) for the mutation? Can yo PCR the locus? If so dHPLC might work for genotyping. Possibly HRM analysis too.