FAQ: Can I Use other primer design tools such as SnapGene for Gibson Assembly, to design primers for NEBuilder HiFi DNA Assembly?

The rules for designing primers are similar for both Gibson Assembly and NEBuilder HiFi DNA Assembly. Any primer designed for Gibson assembly will work with NEBuilder HiFi. However, some primers that work with NEBuilder HiFi may not work with Gibson because unlike the Gibson Assembly Master Mix, NEBuilder HiFi DNA Assembly Master Mix can remove 3' end mismatches. Tools which design "Gibson" primers may not allow the design of primers that take advantage of this ability of NEBuilder HiFi. Though it it is possible to use other tools, NEB recommends using our free tool, NEBuilder Assembly Tool, because it can design primers that consider the vector ends generated by a restriction enzyme digestion.
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    NEBuilder Assembly Tool 2.0 Restriction Enzyme Digest

    This video demonstrates how to use the NEBuilder® Assembly Tool to build a construct using a restriction enzyme digested vector and two PCR-generated inserts.  The tool will help to design PCR primers containing the required overlap sequences.  We will also regenerate one of the restriction enzyme recognition sites.

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    NEBuilder Assembly Tool 2.0 What’s New?

    NEBuilder Assembly Tool can be used to design primers for your NEBuilder HiFi or Gibson Assembly reactions based on entered fragment sequences and the polymerase being used for amplification. This video will highlight some useful updates and the main differences between the two versions.