FAQ: I want to join two fragments, but the overlap sequence is 500bp from the end of the vector. Can NEBuilder HiFi DNA Assembly Master Mix accomplish this?

NEBuilder HiFi DNA Assembly Master Mix can be used to join a DNA fragment 20bp from the end of the vector. To join a fragment at a greater distance from the end, supplement the master mix with 0.1-0.3 units of T5 Exonuclease. Dilute T5 Exonuclease (M0363, 10 units/ul) in 1X CutSmart Buffer. Next, add the diluted enzyme to the assembly reaction along with the DNA 2X master mix. Incubate at 50oC for 1 hour and then transform into high efficientcy competent cells such as NEB 5-alpha or NEB 10-beta. The T5 Exonuclease should be diluted immediately before use and it cannot be store in 1X CutSmart Buffer.