1.Non-specific band at 400 bp, even though by gene size is 650 bp

2.No primer dimer

3.PCR Conditions using right now:

Denaturation - 98 degrees (45 sec), ext.- 98 degrees (30 sec)

Annealing - done for 55 degrees to 61 degrees (Tm for primers was- 58 degrees)- 45 seconds, 35 cycles

Extension- 72 degrees (45 sec.)

Final extension- 72 degrees (15 seconds)