New England Biolabs, M1100S, NEBridge® Ligase Master Mix

Related Categories DNA Ligases,, DNA Assembly, Cloning and Mutagenesis Kits Applications High-throughput cloning and automation solutions,, NEBridge® Golden Gate Assembly Specification Materials Required but not Supplied User-defined DNA fragments User-choice of NEB Type IIS restriction enzymes Competent cells Other materials for transformation FAQ Q: Can Type IIS restriction enzymes from other companies be used with NEBridge Ligase Master Mix? A: Type IIS restriction enzymes from other companies, which have different specifications, have not been tested with NEBridge Ligase Master Mix. Usage recommendations provided are made after extensive optimization. Using non-NEB enzymes is not recommended nor supported. Please follow the reaction conditions found on the product protocol page. Q: Can I assemble PCR amplicons instead of pre-cloned inserts using Golden Gate Assembly protocols and products? A: Yes. For single-insert cloning, you can use unpurified amplicons but please expect reduced performance compared to purified amplicons. For assembly of multiple inserts, amplicons should be purified. For robust purification of amplicons from PCR reaction components, we recommend the Monarch® PCR and DNA Cleanup Kit (NEB #T1030). Q: How does NEBridge Ligase Master Mix perform in Golden Gate Assembly? How does it compare to “home brew” protocols? A: Golden Gate Assembly using NEBridge Ligase Master Mix with seven NEB Type IIS restriction enzymes has been optimized to achieve high efficiency and fidelity, even with complex assemblies of 25 fragments. Depending on the complexity of your assembly reaction and your choice of Type IIS restriction enzyme, you can expect 106-108 cfu per µg vector DNA with a correct assembly rate exceeding 80%. We recognize that many customers prefer to home brew and do this themselves. However, our hope is that by choosing this reagent, we can remove a technical obstacle from your workflow and help you achieve more successful assemblies. Q: I observed high backgrounds with empty vector when using NEBridge Ligase Master Mix. What should I do? A: We suggest performing the transformation immediately after the reaction is done. If you store the reactions at -20°C overnight or for an extensive period, you will need to do another 60°C incubation for 5 minutes right before the transformation. 60°C favors the restriction enzyme activity over T4 DNA Ligase activity and can reduce background by digesting any “re-ligated” vector that still carries the restriction enzyme site. Q: My NEBridge Ligase Master Mix has frozen in my freezer. Is this a problem? A: No. NEBridge Ligase Master Mix generally remains a liquid during storage at -20°C. However, freezer temps vary and if stored below -20°C it may freeze. NEB has performed freeze-thaw testing on this product. After 50 freeze-thaw cycles, over 80% of its original activity was retained. Q: Can NEBridge Ligase Master Mix reactions be inactivated by heating? A: When performing Golden Gate Assembly with this product, there is no need to inactivate it first. While the enzymes will be inactivated if heated extensively, the reaction buffer components may form a complex with the DNA product and negatively impact transformation performance. Q: Can I use the NEBridge Ligase Master Mix reaction to transform electrocompetent cells? A: Yes. Transformation efficiency of electrocompetent cells is generally 10 times higher than chemical competent cells. We have tested direct transformation of the NEBridge reactions using NEB 10-beta electrocompetent E.coli (NEB #C3020). While following the electroporation protocol provided with NEB 10-beta, and using 1 µL of the Golden Gate Assembly reaction with 25 µL of NEB 10-beta electrocompetent cells, we did not experience any problems, such as arcing. Q: Which restriction enzymes are used in Golden Gate Assembly? A: Golden Gate Assembly is a one-tube efficient cloning method based on Type IIS restriction enzymes that cleave outside their recognition sites and leave 3 or 4-base overhangs. BsaI is the most commonly used Type IIS enzyme for Golden Gate Assembly. NEB also offers an engineered high-fidelity version of this enzyme, BsaI-HF®v2, which has the added benefit of being Time-Saver™ qualified (can digest DNA in 5-15 mins or overnight without degradation to DNA) and exhibits dramatically reduced star activity. Other Type IIS enzymes used in Golden Gate Assembly include BsmBI-v2/Esp3I, BbsI/BbsI-HF, PaqCI (AarI isoschizomer with 7 bp recognition sequence), and SapI/BspQI/BspQI-HF (with 7bp recognition sequence and 3bp overhangs). 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: 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.