* Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. To achieve optimal assembly efficiency, design 15-20 bp overlap regions between each fragment.
** To achieve optimal assembly efficiency, design 20-30 bp overlap regions between each fragment with equimolarity of all fragments (suggested: 0.05 pmol each).
† Control reagents are provided for 5 experiments.
†† If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix.