Hey Furkan,

Did you run a gel with the PCR you used to generate your insert?

If I remember correctly the TA kit works by ligating DNA fragments with an overhanging A to the vector (PCRs generate an overhanging A), so it could be possible you ligated two fragments together, but I'd be surprised that that happened with 50% efficiency.

Alternatively in your original PCR you had two PCR products that came out in the clean up. One is longer and ligated into the TA vector.

You could always try a few other test cuts or try for a sequencing read across the two insert sizes. I think the TA vector has M13 universal primers flanking the insertion site.

Good luck,

Chris

Dear Chirostopher,

I loaded my PCR to gel and the size was 1.5 kb. As you said my insert has 3'A overhang and my vector has 5'T. In my case, its obvious that more than 2 insert (most probably 3 ) insert ligated with plasmid but I dont understand how is that possible with 3'A of inserts.

Regards,

While in theory you should not be getting double inserts, strange things often happen in cloning experiments. It usually isn't worth worrying about since you frequently will see them. Just select the right clones and move forward.

Thank you @Micheal I will send constructs in the picture below to screening

@Sundar, I am using taq DNA pol for my ligation protocol. As you now taq DNA is 3' A that is why I did not have problem during the generation of constructs as you can see in the picture below. My concern was about the generation of larger constructs than its expected size as you can see in the picture above. But as dear Micheal Benedik said, generation of larger constructs than expected size is not worth worrying.

I will just wait and see the result of secreening.

Regards,