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1. What is the maximum length of the DNA fragment assembled?

Gibson Assembly Cloning Kit has been used to clone a 15 kb DNA fragment into a 5 4 kb plasmid in E. coli, totaling up to 20 4 kb in length 

2. How many fragments of DNA can I assemble in one reaction? 

The number of DNA segments that can be assembled in one reaction is dependent on the length and sequence of the fragments. We recommend the assembly of five or fewer inserts into a vector in one reaction in order to produce a clone with the correct insert. A strategy involving sequential assembly can be used if all of the fragments cannot be assembled in a single reaction. 

3. Is this method applicable to assemble repetitive sequences?

Yes. However, one must ensure that each DNA fragment includes a unique overlap so that the sequences may anneal and are properly arranged. The repetitive sequence can also be internalized in the first stage of a two-stage assembly strategy. If having repetitive sequences at the ends of each fragment is unavoidable, the correct DNA assembly may be produced, albeit at lower efficiency than other, unintended assemblies. 

4. What are the shortest overlaps that can be used with this assembly method

Productive assembly has been shown for DNA fragments with as little as a 12 bp overlap, however, it depends on the GC content of the overlap. 15 bp overlaps, or more, are recommended for dsDNA assembly with a Tm ≥ 48°C (AT pair = 2°C and GC pair = 4°C).

5. What are the longest overlaps that can be used with this method?

The quantity of 5´ exonuclease in the Gibson Assembly Master Mix and a 15 minute assembly reaction time have been optimized for the assembly of DNA molecules with ≤ 25-bp overlaps. If assembly reaction time is increased to 60 minutes, overlaps up to 40-bp may be used with the Gibson Assembly Cloning Kit.

6. Can ≤ 200 bp dsDNA fragments be assembled by this method?

Yes. For optimal results, use these fragments in ≥ 5-fold excess. 

7. Can I combine and assemble ssDNA oligonucleotides with dsDNA fragments?

Yes. However, the optimal concentration of each oligonucleotide should be determined. As a starting point, we recommend using 45 nM of each oligonucleotide that is less than or equal to twelve 60-base oligonucleotides containing 30-base overlaps. 

8. Can longer or shorter incubation times be used?

Yes. For assembling 2–3 fragments, 15 minutes incubation times are sufficient. For assembling 4–6 fragments, 60 minutes incubation times are recommended. Reaction times less than 15 minutes are generally not recommended. Extended incubation times (up to 4 hours) have been shown to improve assembly efficiencies in some cases. Do not incubate the Gibson Assembly reaction overnight. 

9. Will the reaction work at other temperatures?

The reaction has been optimized at 50°C, but it has been shown to work at temperatures between 40°C and 50°C. 

10. Is it necessary to purify PCR products?

Usually, it’s not necessary. Unpurified PCR products can be used directly, as long as the total volume of PCR products in the Gibson Assembly reaction is 20% or less. If greater amounts of PCR products are used, a column cleanup kit is sufficient. 

11. Is it necessary to inactivate restriction enzymes after vector digestion?

Generally, inactivation is not necessary, but in some cases it might increase the transformation efficiency. If the insert also carries the restriction site that was used to linearize the vector it is necessary to heat inactivate the restriction enzyme before mixing the linearized vector with the insert in Gibson Assembly. If a heat-resistant restriction enzyme was used to linearize the vector, then vector should be purified by phenol-chloroform extraction or extracted from agarose gel after electrophoresis, before coming into contact with the insert.

12. I would like to produce overlapping dsDNA fragments by PCR. Do I need to use PAGE or HPLC purified PCR primers?

No. Standard, desalted primers are enough 

13. I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do I need to use oligonucleotides purified by PAGE or HPLC?

No. Standard, desalted primers may be used. 

14. Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e. CACCACCACCACCAC)?

No, you must flank the His-tag sequence on both sides with at least 2 nucleotides, that are not part of the His-tag repeating sequence. you should avoid repeating sequences at the end of an overlap 

15. Can I PCR-amplify the assembled product?

Yes. The assembled DNA molecule is covalently joined and may be PCR-amplified. Additionally, if the final product is a closed circular DNA molecule, it may be used as a template in rolling-circle amplification (RCA) 

16. The Gibson Assembly Master Mix control reaction is not giving me any colonies. Why?

Our testing indicates that the choice of competent cells is critical. We recommend the use of high efficiency chemically competent cells such as NEB 5-alpha Competent E. coli (High Efficiency) (NEB #C2987). The reaction can be added directly to the cells without any dilution, although further dilution of the reaction mix may improve transformation efficiency. However, when using high efficiency chemically competent cells from some other vendors, if you did not get any colonies, we recommend a 1:4 dilution of the reaction prior to transformation. For transformation into all high efficiency electro competent cells, including NEB's, we recommend a 1:3 dilution of the reaction 

17. What should I do if my assembly reaction yields no colonies, a small number of colonies, or clones with the incorrect insert size following transformation into E. coli?

Assemble and transform the positive control provided with the Gibson Assembly Master Mix (see page 11,12) Successful assembly of a positive control will demonstrate that the assembly mixture is functional and the transformation conditions are suitable. 

Analyze the reaction on an agarose gel. An efficient assembly reaction will show assembled products of the correct size and the disappearance of fragments.

Check the primer design of the overlapping DNA fragments to ensure that there is sufficient overlap to facilitate assembly.

Consider whether the cloned insert may be toxic to E. coli and a low-copy vector, such as a BAC, should be used. Because the assembled product is a covalently closed molecule, it may be alternatively amplified by PCR or RCA.

18. How can I reduce the number of vector-only background colonies?

To significantly reduce the background of unwanted vector-only colonies, the vector should be a PCR product rather than a restriction fragment. If background continues to be a problem, the PCR-amplified vector can be treated with DpnI to remove the template carry-over, if applicable, extracted from an agarose gel following electrophoresis 

19. What type of competent cells are suitable for transformation of DNA constructs created using Gibson Assembly?

The resulting DNA constructs are compatible with most E. coli competent cells NEB recommends using NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987) If the assembled products are larger than 10 kb, NEB recommends using NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019) or NEB 10-beta Electro competent E. coli (NEB #C3020)  

20. Can I use electroporation instead of chemical transformation?

Yes, but it is necessary to dilute the Gibson Assembly reaction product 3-fold, and use only 1 µl for electroporation. Note: The cells provided with this kit are chemically competent 

21. Are there any differences between the requirements for 2–3 fragment assemblies versus 4–6?

The major differences between the two are the length of overlapping sequences between the adjacent fragments and the incubation time of the assembly reaction. The 15 minute assembly reaction protocol is recommended for assembly of 2–3 fragments that are flanked by 15–25 nt overlaps. The 1-hour assembly protocol is recommended for the assembly of up to 6 fragments, flanked by 20–80 nt overlaps. The total amount of DNA in a 4–6 fragment assembly is also higher than a 2–3 fragment assembly.