Hi

I used DNA template having concentration of 31ng for my mutation PCR, but no mutation occur after sequencing.

my PCR protocol is as follows                                         Temp                   time

1. ddH2O               = 36ul                                             1. 94C                      4 Minutes

2. 10*buffer            = 6ul                                               2. 94C                      45 Seconds  

3. dNTP                 = 6ul                                               3.  55C                     30 Seconds

4.  primerF             = 6ul                                               4.  72C                     7 Minutes

5.  primerR            = 6ul                                                5.  72C                     10 Minutes

6.  Template         = 1ul                                                 6.  4C                        Infinity

7  taq polymerase =0.5 ul

    total volume = 60 ul for two reaction

the PCR product was  run on gel electrophoresis, which give us good bands, then we digested the product by dpn1 and incubated for two hours , transferred to DH10b competent cell, make single colony culture, extract the plasmid DNA, find the concentration by NANODROP and finally cut the plasmid by Xho1 and Nde1 to confirm our gene by gel electrophoresis . after confirmation of our gene in the plasmid we send the sample to company for sequencing, but after result no mutation occur .

please suggest me best way to get my desire site mutation and also let me know that the protocol which I am using is ok for mutation or not ?