What is your template and PCR conditions? Are you using DMSO 3-5% in PCR reaction? Have you checked if the primers with overhang are binding elsewhere in the template? Please answer these to troubleshoot your PCR.

The 120 bp DNA is the downstream region of a 2.2 kb gene cloned in pet20b vector. This clone is used as a template. I have tried using a range of DMSO conc. from 2-10% but then I get rid of the primer dimers but do not get the desired amplicon. Also the overhang does not bind to any other region in the template nor has any complimentarity with the desired product DNA. Other reagents used for the PCR are working fine too. 

Dear Sumedha,

I have gone through your problem. Have you tried touch-down PCR for your fragment 3?. Normally, when the Ta value of fw and rv primers varies much, say for example more than 10deg C (as in your case too), then normal conventional PCR will not work that effectively because the annealing temperature that you keep will not favor one of the primer to bind.

In this situation, you can perform

1) TD-PCR keeping Ta range between 82 and 70.

2) Use 5%DMSO with 1M Betaine combo.

3) Use Sapphire or Emerald GT PCR master mix from TaKaRa, Japan.

Most of the time i get good amplicons with TD-PCR + 5%DMSO+1M Betaine+either of these master mix.

All the best

With best regards,

S.Rajkumar

Thanks for the information. I have done similar experiments to generate internal deletion. Q5 polymerase for me was quite effective for difficult PCRs (https://www.neb.com/products/m0491-q5-high-fidelity-dna-polymerase) followed by Gibson assembly kit (https://www.neb.com/products/e2611-gibson-assembly-master-mix). I hope it works for you as well. Good luck

Thanks for this. I will give it a try.