Hi Roba Alatawy,

I think one of your primer's complementary binding site was lost because of the transposon insertion, that is why you are not getting the amplification product. 

In this condition I would blast the primer sequence with the mutated gene to check whether the complementary sequence is intact or not. If the primer binding site is destroyed because of the insertion, you have to design a new pair of primer.

Hope it will help,

Best,

Biswaranjan Pradhan

Ph.D Student, SBS,

NISER Bhubaneswar

Hi Roba Alatawy,

I think one of your primer's complementary binding site was lost because of the transposon insertion, that is why you are not getting the amplification product. 

In this condition I would blast the primer sequence with the mutated gene to check whether the complementary sequence is intact or not. If the primer binding site is destroyed because of the insertion, you have to design a new pair of primer.

Hope it will help,

Best,

Biswaranjan Pradhan

Ph.D Student, SBS,

NISER Bhubaneswar

Hi Roba,

One possibility is indicated above....In addition, make sure that you are keeping the 'elongation step' of PCR sufficiently long enough to amplify the amplicon with the insertion....If the site is lost ...you can check by PCR using the original primers in combination with two primers designed in the transposon

bye

Thank you so much Ajay and Biswaranjn. What you said here make sense. I will design new primers pair and will let you know of the result.