FAQ: How can I improve the efficiency of very complex assembly reactions when using NEBuilder HiFi DNA Assembly Master Mix?

A very complex assembly reaction can contain 6 or more DNA fragments, including unpurified restriction enzyme digestion products.  For example, you may be using an insert that has been cut out of a plasmid but not purified, so that there are two DNA fragments: the insert and the original vector. Only the insert fragment will assemble with other fragments.  The efficiency of these very complex assembly reactions can be increased by incubating the reactions at 45°C instead of 50°C.  We recommend using 0.05pmol of each digested plasmid, with a maximum of 0.5pmol of DNA in the reaction.