I have to join four fragments of sizes 1.2 (A), 2.5 (B), 1.9(C) and 0.7kb (D) each having an overlapping region of around 50bp with Tm >80°C. I successfully amplified individual fragments ( as a single band) and purified them with the PCR purification kit. This whole exercise didn't take enough time.
Now comes the hard task of joining;
I performed overlap extension PCR for 10 cyces using equimolar conc. of fragment A and B in 25µl reaction volume using phusion high fidelity polymerase. After 10 cycles, I added 25µl fresh master mix containing Forward-A and Reverse-B primers and allowed the amplification to continue for another 30 cycles. Since Tm of overlapping region was >72°C, I used 72°C for annealing as well as extension termperatures.
The result I get every time is "smearing" . I tried different MgCl2 conc. as well as different polymerases however, smearing pattern is consistently reproduced
Same is the case with C and D.
Could you please give me any suggestion that can help me getting rid of this smearing pattern.
Thanks in advance.