I am doing site-specific mutation experiment. But my experiment did not work. I have try to change many factors to fix it. But the effect is not apparent. Below is what I have done:
The site-specific mutation did not work. The way to fix it:
Possible cause and suggested solution
PCR system did not work? No, positive control work.
Competent cells is not good? No, the control result showed that it is better than normal commercial competent cells
Template is wrong? No, it was sequenced and the ORF is right.
PCR condition is not proper? Probably but maybe not. I have try to increase the denature Tm to 98 oC, it did not work.
Primers are not good? Probably but maybe not. I use the same PCR system and different annealing temperature to amplify my protein truncated using Protein_F and mutant_R. It work!!!at different annealing temp. form 40-60 oC Which showed that the mutation primers can bind template. And the enzyme system can amplify the gene properly.
DO you have any suggestion? Please tell me.
Thank you in advance.