New England Biolabs, E1602S, NEBridge® Golden Gate Assembly Kit (BsmBI-v2)

Related Categories DNA Assembly, Cloning and Mutagenesis Kits Applications DNA Assembly and Cloning,, High-throughput cloning and automation solutions,, NEBridge® Golden Gate Assembly Specification Materials Required but not Supplied User-defined inserts Competent cells Other materials for transformation FAQ Q: What is the mechanism for Golden Gate Assembly? A: Assembly utilizes two simultaneous enzymatic activities in a single reaction, Type IIS restriction endonuclease digestion and T4 DNA Ligase ligation. With optimized buffer components and enzyme ratios, a single reaction containing a destination plasmid and inserts (PCR amplicons or precloned) will result in ligation of inserts in the correct order and the accumulation of assembled product over time. The final assembly has none of the chosen Type IIS recognition sites, rendering the assembly inert to further digestion. For more information, view our online tutorial at www.neb.com/goldengate. Q: Which kit from NEB should I use for Golden Gate Assembly—the BsmBI-v2 kit (NEB #E1602) or the original BsaI-HFv2 kit (NEB #E1601)? A: It depends on whether there are any internal sites for these enzymes in your insert sequences. Since internal sites need to be eliminated by site-directed mutagenesis, choose the kit based on the Type IIS restriction enzyme that has no, or the fewest, sites in your insert sequences. If your sequences have neither BsmBI nor BsaI sites, the BsmBI-v2 kit (NEB #E1602) would be the best choice as it supports the highest complex assembly performance yet developed at NEB both in terms of efficiency (number of transformants) and fidelity (% correct assemblies). However, both kits support complex 24 fragment assemblies. You also have the option to use NEBridge Ligase Master Mix (NEB #M1100) which is designed for use with any NEB Type IIS restriction enzyme. Q: What if there are internal BsaI and BsmBI sites in my insert sequences? A: Either use site-directed mutagenesis to eliminate the internal sites beforehand, or utilize the internal sites as junctions between fragments using primers to introduce the silent mutations into the sequence simultaneously during part generation. Or screen your sequences for the absence of other Type IIS restriction sites that could allow an alternative Type IIS restriction enzyme to be used such as BbsI, SapI/BspQI or BtgZI (building your assembly reactions using individual restriction enzyme and T4 DNA Ligase stocks), or consider another assembly approach such as NEBuilder® HiFi DNA Assembly if the assembly will involve 5 or less inserts. Please refer to our Golden Gate Domestication Tutorial. Q: The Type IIS restriction enzyme used in this kit is BsmBI-v2. How does it compare to the original BsmBI or Esp3I? A: While all 3 can perform complex (24-fragment) assemblies, BsmBI-v2 had the highest efficiency (number of transformants) and fidelity (% correctly assembled constructs) levels of the three. These comparison experiments used the individually optimized temperatures for each enzyme, 42°C for BsmBI and BsmBI-v2, and 37°C for Esp3I. Q: How does the performance of this kit compare to “home brews” built with individual Type IIS restriction enzymes and T4 DNA Ligase components? A: Building assembly reactions with separate enzyme components available at concentrations appropriate for standard digestion of DNA are limited by the need to keep glycerol levels in the reactions ideally at or less than 10%. These “home brew” assemblies will work, but will not match the efficiency and fidelity achieved with this kit that features higher enzyme levels. Q: What affects the efficiency of Golden Gate Assembly? A: Single insert cloning is significantly more efficient than multiple insert cloning. Assembly efficiency decreases as the number of fragments increases. The presence of repetitive sequences in an insert will also decrease efficiency. For inserts < 250 bp or > 3 kb, precloning will increase efficiency. Lastly, the normal restrictions on overall plasmid size that allow stable maintenance in E. coli apply to Golden Gate Assemblies. Efficiencies are highest with assembled product plasmid constructs ~ 10-12 kb. Larger sized completed assemblies can be made but will require larger numbers of colonies to be screened for the correct full length assembled products. For experimental examples of complexity vs. efficiency, refer to the Golden Gate Assembly Technical Note. Q: Why do many of the published Golden Gate Assembly articles feature precloned inserts as opposed to inserts generated by PCR? A: Precloned inserts allow stable storage of inserts while using amplicon inserts saves time. Stable storage of amplicon inserts is important and is best in a buffered solution. Single insert cloning/assemblies can use unpurified amplicons but will result in lower performance than if purified, while multiple insert amplicons should be purified, for example, by spin column protocols. We recommend the Monarch® PCR and DNA Cleanup Kit (NEB #T1030). For long term storage at –20°C, store DNA in 10 mM Tris (pH 8.5), 1 mM EDTA (TE) or short-term storage in 10 mM Tris (pH 8.5), 0.1 mM EDTA (modified TE). EDTA at these levels will not significantly lower the 10 mM MgCl2 present in the T4 DNA Ligase Buffer used for assembly reactions. Q: Using purified amplicons directly without precloning seems much easier, but is the assembly efficiency decreased? A: No. While in general DNA is more stable in circular form than in linear form due to the absence of free ends, amplicons are a viable and easy way to build assemblies as long as they have been purified and are stored in the appropriate buffer (see FAQ). The suggested 2:1 molar ratio of amplicon inserts : destination vector brings the assembly efficiency to that of precloned inserts (using 75 ng of each plasmid) for most assemblies. Q: Can PCR amplicons be used directly in assembly reactions without purification? A: Yes, as long as insert volume is 1 µl or less and single insert (cloning) is being done. Although efficiencies will be decreased. Most Type IIS restriction enzymes used for Golden Gate Assembly generate 5´-four base overhangs that can be filled-in by the carryover DNA polymerase used in PCR when using unpurified amplicons, producing blunt ends. This will lead to nonspecific assembly. For single insert cloning/assembly, the ligase successfully competes with the carryover DNA polymerase such that unpurified PCR amplicon inserts can be used but will result in lower assembly performance. For multiple insert Golden Gate assemblies, purify the amplicons and if non-specific products are present, optimize the PCR or gel purify. Q: Why is Golden Gate also used for single insert cloning? A: While Golden Gate is normally used for insert assemblies of 5–10 or more fragments, it also allows easy and highly efficient cloning of single inserts following the provided directions. Golden Gate can also be used with diverse single insert populations for library preparations and directed evolution requiring multiple site mutagenesis. Q: Why do assembly reactions end with a 5 minute, 60°C incubation step? A: The final incubation step at 60°C favors Type IIS restriction enzyme cutting, in the absence of DNA ligation. Digesting any uncut or cut/religated destination plasmid still present in the assembly reactions reduces background. Q: How can I minimize PCR-generated errors in my amplicon inserts? A: Use a high-fidelity DNA polymerase and avoid over-amplification. We recommend Q5® High-Fidelity DNA Polymerase formulations for maximal fidelity (NEB #M0491, #M0493), which is also available in Master Mix format (NEB #M0492, #M0494). Also, use the minimum number of cycles required to generate the amount of DNA required for assembly; this is usually 20 cycles or less. Q: What is an appropriate negative control for Golden Gate Assembly? A: Golden Gate assembly protocols do not usually call for a negative control. However if desired, a “no insert(s) added” reaction can be used. Q: How many cycles are optimal? A: Our enhanced enzyme stability allows more cycles than the traditional 30 cycles if larger numbers of transformants are desirable for complex assemblies or single insert library generation. For both our original BsaI-HFv2-based assembly kit and our BsmBI-v2-based assembly kit, efficiency increases dramatically from 30 cycles to 60-65 cycles, with no loss of fidelity. Q: Can I use other competent E. coli strains than NEB 10-beta? Can I use subcloning efficiency cells? A: Yes, other cell strains can be used, but large assemblies will require strains known to maintain large plasmid stability, such as NEB 10-beta competent E. coli (High Efficiency, NEB #C3019) or NEB Stable Competent E. coli (High Efficiency, NEB #C3040). NEB Stable Competent E. coli are also recommended for inserts containing repeat/unstable elements. For smaller assemblies other strains such as NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987), NEB Turbo Competent E. coli (High Efficiency, NEB #C2984) or NEB T7 Express Competent E. coli (High Efficiency, NEB #C2566) can also be used. Subcloning efficiency cells will result in lower transformation levels and should not be used for multi-component assemblies. Q: How can I access pGGAselect as a GenBank or FASTA file? A: Please visit DNA Sequences and Maps Tool for pGGAselect FASTA or GenBank formatted sequence files, and its plasmid map. Q: How many base pairs should my amplicon inserts have flanking the Type IIS restriction site? A: Amplicon inserts must posses 5' flanking bases and Type IIS restriction sites at both ends of the amplicon in the proper orientation. We recommend adding 6 flanking bases at the 5' ends of the primers for optimal Type IIS restriction enzyme binding, cleavage, and overall Golden Gate Assembly efficiency. This 6 base pair recommendation supersedes other known end length preferences for the Type IIS restriction enzymes due to Golden Gate Assembly protocols requiring maximum enzyme activity for efficient assembly.