Recommended Screening Protocols for Using NEB Golden Gate Assembly Mix (E1600)
- Colony PCR screening using an appropriate DNA polymerase for amplification of the insert region. While many DNA polymerases are suitable for colony PCR, OneTaq ® DNA Polymerase (NEB #M0480) or OneTaq Hot Start DNA Polymerase (NEB #M0481) are recommended. Taq DNA Polymerase (NEB #M0267) can also be used. For PCR of larger assemblies, LongAmp® Taq DNA Polymerase (NEB #M0323) or LongAmp Hot Start Taq DNA Polymerase (NEB #M0534) are strongly recommended.
- Preparing plasmid mini-preps using the Monarch Plasmid Miniprep Kit (NEB #T1010 ) and mapping by using appropriate restriction endonucleases to confirm the correct assembly.
Regardless of the screening protocol used, the correct assembly of insert(s) should always be confirmed by sequencing of the plasmid construct across the 4 base junctions and inserts.