FAQ: I would like to use colony PCR to screen my transformants and sequence my assembly. Are there any recommended sequencing or colony PCR primers?

Analysis primers are not included with the Golden Gate Assembly Mix product. We recommend the following oligos for pGGA-based assemblies be custom ordered through any oligo synthesis provider: Forward (CW) primer 54 bp upstream from assembly point: 5´-CTGCAGGAAGGTTTAAACGCATTTAGG-3´ Reverse (CCW) primer 51 bp downstream from assembly point: 5´-TAATACGACTCACTATAGGGAGACGC-3´ Note that larger assemblies will require internal assembly-specific primers to verify assembly sequence. As an alternative, the cloning analysis forward primer featured in the NEB PCR Cloning Kit can be used, but is located further upstream of the assembly point. Also many commercially available T7 promoter oligos can be used from the reverse direction.