New England Biolabs, E2611S, Gibson Assembly® Master Mix

Gibson Assembly Master Mix has been reformulated with components containing Recombinant Albumin (rAlbumin) beginning with Lot #10229799. Related Categories DNA Assembly, Cloning and Mutagenesis Kits Applications Gibson Assembly Specification Materials Required but not Supplied Recommended DNA polymerase options for PCR Q5® High-Fidelity DNA Polymerase (NEB #M0491) Q5 Hot Start Flex DNA Polymerase (NEB #M0493) Q5 Hot Start Flex 2X Master Mix (NEB #M0494) LB (Luria-Bertani) plates with appropriate antibiotic SOC Outgrowth Medium (NEB #B9020) Competent cell options for transformation NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987) NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019) (Recommended for assembled products greater than 10 kb) NEB 10-beta Electrocompetent E. coli (NEB #C3020) NEB® Stable Competent E. coli (High Efficiency) (NEB #C3040) Storage Notes Store at -20°C. Thaw, vortex thoroughly before use and keep on ice. FAQ Q: I am not sure whether to choose NEBuilder HiFi DNA Assembly or NEB Gibson Assembly? How are the products different? A: NEBuilder HiFi DNA Assembly offers several advantages over NEB Gibson Assembly. NEBuilder HiFi utilizes a proprietary higher fidelity polymerase, which results in less screening/re-sequencing of constructs and virtually error-free, high-fidelity assembly. This feature also enables NEBuilder to be used in certain applications that NEB Gibson Assembly cannot be used for: NEBuilder HiFi removes 5´- and 3´-end mismatches, and can be used in successive rounds of assembly. This saves time by avoiding time-consuming PCR amplification steps. NEBuilder HiFi can bridge two ds-fragments with a synthetic ssDNA oligo for simple and fast construction (e.g., linker insertion or gRNA library). For more detailed information and data, please visit NEBuilderHifi.com Q: What are the advantages of this method compared to traditional cloning methods? A: Gibson Assembly allows insertion of one or more DNA fragments into virtually any position of the linearized vector and does not rely on the presence of restriction sites within a particular sequence to be synthesized or cloned. Therefore, the user has complete control over what is assembled and insertion of unwanted additional sequence, often used to facilitate the manipulation of multiple DNA sequences, can be avoided. Furthermore, the Gibson Assembly method is fast relative to standard restriction enzyme-based cloning. Lastly, a greater number of DNA fragments can be joined in a single reaction with greater efficiency than conventional methods. Q: How large a DNA fragment can I assemble? A: Gibson Assembly Cloning Kit (containing NEB 5-alpha Competent E. coli ) has been used to successfully clone a 15 kb DNA fragment into a 5.4 kb plasmid in E. coli, totaling up to 20.4 kb in length. However, as a general rule for assembled products greater than 10 kb, NEB recommends NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019) or NEB 10-beta Electrocompetent E. coli (NEB #C3020). Q: How many fragments of DNA can be assembled in one reaction? A: The number of DNA segments that can be assembled in one reaction is dependent on the length and sequence of the fragments. Gibson Assembly has been used to efficiently assemble up to twelve 0.4 kb inserts into a vector at one time. However, 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. Alternatively, consider Golden Gate Assembly for assemblies of >6 fragments. Q: What are the shortest overlaps that can be used with this assembly method? A: Productive assembly has been achieved for DNA fragments with as little as a 12 bp overlap, however, it depends on the GC content of the overlap. We recommend using at least 15 bp overlaps, or more, for dsDNA assembly with a Tm ≥ 48°C (AT pair = 2°C and GC pair = 4°C). Increasing the length of overlap between fragments also reduces the amount of DNA needed for assembly. Q: What are the longest overlaps that can be used with this method? A: The quantity of exonuclease in the Gibson Assembly Master Mix has been optimized for the assembly of DNA molecules with ≤ 100-bp overlaps. Q: Can ≤ 200 bp dsDNA fragments be assembled by this method? A: Yes. For optimal results use each smaller fragment at ≥ 5-fold molar excess than the vector (5:1 insert: vector) and keep the total amount of DNA in the Gibson reaction to 0.02 -0.5 pmoles. Q: Can ssDNA oligonucleotides be combined and assembled with dsDNA fragments? A: 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. Q: Can longer or shorter incubation times be used? A: Yes. For assembling 2-3 fragments, 15 minute incubation times are sufficient. For assembling 4-6 fragments, 60 minute 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 reaction overnight. Q: Will the reaction work at other temperatures? A: The reaction has been optimized at 50°C, but it has been shown to work between 40°C and 50°C. Q: Is it necessary to inactivate restriction enzymes after vector digestion? A: Inactivation of restriction endonucleases is generally 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 DNA columns, phenol-chloroform extraction or extracted from agarose gel after electrophoresis, before coming into contact with the insert. Q: I would like to produce overlapping dsDNA fragments by PCR. Do I need to use PCR primers that have been purified by PAGE or HPLC? A: No. Standard, desalted primers may be used. Q: I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do I need to use oligonucleotides that have been purified by PAGE or HPLC? A: No. Standard, desalted primers may be used. For more information, please download the application note, Construction of an sgRNA-Cas9 expression vector via single-stranded DNA oligo bridging of double-stranded DNA fragments. Q: Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e. CACCACCACCACCAC)? A: No, after the His-tag, you must include at least 3 nucleotides, that are not part of the His-tag repeating sequence. Avoid repeating sequences at the end of an overlap. Q: Can you PCR-amplify the assembled product? A: 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). Q: 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? A: Assemble and transform the positive control provided with the Gibson Assembly Master Mix. 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. 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 electrocompetent cells, including NEB's, we recommend a 1:3 dilution of the reaction. Q: How can I reduce the number of vector-only background colonies? A: 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. Q: What type of competent cells are suitable for transformation of DNA constructs created using Gibson Assembly? A: 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 Electrocompetent E. coli (NEB #C3020). If the assembled genes contain repetitive sequences, NEB Stable Competent E. coli (NEB #C3040) should be used. Q: Can I use electroporation instead of chemical transformation? A: 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. Q: Are there any differences between the Gibson Assembly Master Mix (NEB #E2611) and Gibson Assembly Master Mix included in the Gibson Assembly Cloning Kit (NEB #E5510)? A: No, the master mix is the same in both kits. The Gibson Assembly Cloning Kit (NEB #E5510) includes NEB 5-alpha chemically competent E. coli. Q: Are there any differences between the requirements for 2-3 fragment assemblies versus 4–6? A: 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. Q: The Gibson Assembly Master Mix control reaction is not giving me any colonies. Why? A: 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 electrocompetent cells, including NEB's, we recommend a 1:3 dilution of the reaction. Q: When using a polymerase that doesn't contain a 3'-5' exonuclease activity (such as Taq DNA Polymerase) to amplify fragments to be used in a Gibson Assembly reaction, should I be concerned about the potential 3' mismatch generated by the addition of a non-templated nucleotide? A: This type of 3' mismatch can affect the efficiency of the assembly, especially when attempting to assemble a high number of fragments (4-6). In those cases, it may be necessary to screen more colonies to obtain the correctly assembled clone. To prevent this, we recommend the use of polymerases that produce blunt-ended amplicons, such as Q5 High-Fidelity DNA Polymerase . Q: Is storing Gibson Assembly Master Mix at -80°C harmful? A: NEB recommends storing the Gibson Assembly Master Mix at -20°C, however the Master Mix is stable at -80°C. When you use the Master Mix for the first time, please transfer it to -20°C for future storage. Q: I would like to use NEBuilder but am concerned about user data privacy. How does NEB handle the information that I enter into NEBuilder? A: NEBuilder is sensitive to user data privacy. At this point, user sequences *do not* leave the client browser. All of the processing is done within the client browser. NEB servers are contacted to: 1. Download the page and required code to the browser 2. Retrieve help files, manuals, and images 3. Retrieve sample vector and/or insert sequences No user data is transferred out of the browser across your firewall to NEB's servers. This can be verified by disabling your internet connection to the browser after the page has loaded. While you will not have the convenience of getting vector sequences, the rest of the application should work fine and produce the desired primers. NEBuilder is not open-sourced at this time, nor is it available in a standalone desktop version. Q: Can I Use other primer design tools such as SnapGene for Gibson Assembly, to design primers for NEBuilder HiFi DNA Assembly? A: The rules for designing primers are similar for both Gibson Assembly and NEBuilder HiFi DNA Assembly. Any primer designed for Gibson assembly will work with NEBuilder HiFi. However, some primers that work with NEBuilder HiFi may not work with Gibson because unlike the Gibson Assembly Master Mix, NEBuilder HiFi DNA Assembly Master Mix can remove 3' end mismatches. Tools which design "Gibson" primers may not allow the design of primers that take advantage of this ability of NEBuilder HiFi. Though it it is possible to use other tools, NEB recommends using our free tool, NEBuilder Assembly Tool, because it can design primers that consider the vector ends generated by a restriction enzyme digestion. Q: What antibiotic resistance is encoded in the NEBuilder Positive Control (assembly control)? A: The NEBuilder Positive control is a single insert in a pUC19 plasmid which is Ampicillin resistant. If your Assembly Mix is working you will get colonies on an ampicillin plate using the NEBuilder Positive Control.