Hi Elar,

This situation is quite common and can happen due to a number of reasons including failed restriction digestion or failed ligation reaction. Ligation reaction PCR will show bands as it contains large amounts of insert. since your objective is to do Sanger sequencing, I suggest you to do the original PCR again with Taq Polymerase with a final extension step @ 72C for 20-30 mins , then ligate your amplicon into any T/U-A cloning kit (eg. Thermo InsTA cloning kit or Qiagen pDrive cloning kit) and then do sequencing with vector primers (m13F & m13R).

best wishes.

Sibnarayan Datta, is it 20 to 30 Minutes or Seconds!

Elar, I am referring to the final extension step, i.e., after the PCR cycles, before final hold. This is 20-30 minutes to add more of A bases at the 3' ends of your amplicons. This will enhance T/A cloning efficiency. And do NOT use a proofreading polymerase, just use a good quality conventional Taq polymerase. Everything will be ok if you follow these. Just go ahead, best of luck .

SD

Elar, I am referring to the final extension step, i.e., after the PCR cycles, before final hold. This is 20-30 minutes to add more of A bases at the 3' ends of your amplicons. This will enhance T/A cloning efficiency. And do NOT use a proofreading polymerase, just use a good quality conventional Taq polymerase. Everything will be ok if you follow these. Just go ahead, best of luck .

SD

Sibnarayan , Thank you.

I will try, however, I use Pfu, Takara, Taq. I hope nest time get a good result.

Best,

Elar