Hi Priya, 

Forward sequencing might not work because of a primer issue (assuming you repeated the sequencing and it definitely doesnt work). The primer might have degraded for example. It happened to me before. Nevertheless, the sequencing product wont be more than 400bp so you do need to design primers closer to the site of mutation. From what you said, I understand you did that already - designed a reverse primer. Just to confirm, was this primer inside the gene ? If the sequencing result you got from that primer you designed matches your vector then perhaps you designed the primer the wrong way. Try designing both a forward and reverse primer close to the mutation site (maybe 50-100bp away from the site).   

Hope this helps.

Constantia

Hi Constantia,

Many thanks for your suggestions!

The forward primer that I had used looks good. I had sent the wild type plasmid for sequencing using the same forward primer (as a positive control) and the sequencing results are fine. 

Definitely, I will design both forward and reverse primers close to the mutation site and will check if that works.

Best Regards,

Priya

i might add use the PCR product of forward and reverse primer that you will design and then make it template and do another round of PCR, then gel elute or PCR purify and use it as template for cycle sequencing for both forward and reverse primers separately 

First,I come across the same problem,but my may overcome it and finally succed,if your produce is very faint ,do not worry,just deal with DpnI and without purify,transform directly,the second method ,if you want to overcome the faint issue,you re-design your PCR primers,but different ,on the side of each primers,it should longer,simply say,except the center mutant area,each 3'-terminal shuold longer