F1-X™ Next-Gen Gibson Assembly®

1-Step, seamless cloning without restriction site dependence; scarless junctions with no unwanted sequences; versatile for genes, plasmids, pathways, and whole genomes; direct use for transformation, PCR, or rolling circle amplification.

Yes. The covalently joined DNA can be used as a PCR template or for rolling circle amplification without purification. As a starting point, we recommend testing 1 µL of F1-X™ reaction in a 20 µL PCR or rolling circle amplification reaction following manufacturer guidelines.

Yes. Use 45 nM as starting concentration for each oligonucleotide. Oligonucleotides >90 bases may have interfering secondary structures.

Yes. F1-X™ can assemble multiple linear fragments into complete plasmids, including entire constructs from synthetic DNA. Note that the orientation of overlap design dictates the final construct sequence.

For individual fragments: up to 32 kb. For total construct size: up to 100 kb. Larger constructs are possible with multi-stage assembly.

Up to 12 fragments is recommended for single-stage assembly, and we have verified up to 14 fragments in a single reaction. It is critical that overlaps are 40 bp or more for multi-fragment assemblies. More fragments may be possible with multi-stage approaches.

Yes. Design fragments with repetitive sequences internalized rather than at overlap regions, or use longer overlaps for uniqueness.

Yes. F1-X™ handles fragments as small as 100 bp. Use ≥5-fold molar excess for optimal results.

No. Vectors can be linearized by restriction digest. All end types (blunt, 5′ overhang, 3′ overhang) are compatible. Vectors can also be prepared synthetically as gene blocks or fragments.

We recommend 40 bp as a starting point for all assemblies. For simple assemblies of 2–3 fragments, as little as 20 bp overlaps can work. For complex constructs of 4+ fragments, use 40+ bp.

Store at -20°C in a stable temperature location (not a frost-free freezer). Limit to 5 freeze-thaw cycles or aliquot for frequent use

Use the amounts specified in the protocol section. Lower concentrations can be tested if materials are limiting.

Use a minimum 15 minutes for simple assemblies; for more complex assemblies, we recommend starting with a 1 hour assembly time, then optimizing for shorter reaction times down to 15 minutes as allowed. We have seen success with multi-fragment assemblies (including the 4-fragment positive control and a 9-fragment control) down to 15-minute reaction times, but 60 minutes is a conservative place to start.

We recommend 50°C as a starting point for all assembly reactions. F1-X™ contains a proprietary blend of high-fidelity enzymes that are active between 50°C–56°C. If your construct requires additional optimization for enhanced annealing stringency, run the assembly at 53°C–56°C and extend the reaction time between 1–2 hours.

Yes. Residual restriction enzyme can cause star activity and DNA degradation. Is gel purification necessary? It is not required if PCR yields >80% full-length product. Low molecular weight contaminants caused by off-target amplification in PCR have a detrimental effect on cloning efficiency.

Sequence entire insert plus ~500 bp of vector at junctions. If PCR was used for vector preparation, consider sequencing entire construct using NGS or Sanger sequencing. We recommend consulting with your sequencing vendor to find the right workflow for your needs.

No, this is normal. Agarose gel electrophoresis is a qualitative, construct-dependent indicator of assembly success. Gibson Assembly reactions often produce high molecular weight species that appear as laddering rather than discrete bands. Compare your assembly reaction to a no-assembly control and look for reduced substrate bands and higher molecular weight products. For definitive confirmation, use transformation as your primary success metric — colony count and sequencing verification provide definitive confirmation of assembly success.