I have been trying to join 3 fragments (3kb, 2kb and 1kb respectively) in the last days using the DJ PCR protocol but to no avail. My 2nd PCR annealing temperature was at 57°degree for 10 min and extension time of 6 min. Anyone with such experience before? Alternatively, I have used the NEB builder mix which only worked for the 2 PCR fragments omitting the middle fragments which contains my marker.