New England Biolabs, E2623S, NEBuilder® HiFi DNA Assembly Bundle for Large Fragments

NEBuilder HiFi DNA Assembly Master Mix has been reformulated with components containing Recombinant Albumin (rAlbumin) beginning with Lot #10238675. Related Categories DNA Assembly, Cloning and Mutagenesis Kits Applications NEBuilder® HiFi DNA Assembly Specification Materials Required but not Supplied Nuclease-free Water (NEB #B1500) 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: NEBuilder HiFi DNA 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. 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 NEBuilder HiFi DNA 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: What is the largest single fragment that has been assembled with NEBuilder HiFi DNA Assembly Master Mix? A: NEBuilder HiFi DNA Assembly Master Mix has been used to clone a construct 22.7 kb in length. For assembled products greater than 15 kb, NEB recommends NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019) which are included in the NEBuilder HiFi DNA Assembly Bundle for Large Fragments (NEB #E2623). Alternatively, you can use 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. NEBuilder HiFi DNA Assembly Master Mix has been used to efficiently assemble up to eleven, 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: Can ≤ 200 bp dsDNA fragments be assembled by this method? A: Yes. For optimal results, use these fragments in ≥ 5-fold excess. Q: Can multiple ssDNA oligonucleotides be 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. 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 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 purify PCR products when doing DNA assemblies using either NEBuilder HiFi DNA Assembly Master Mix or Gibson Assembly Master Mix? A: Purification of PCR products is generally not necessary. You can use unpurified PCR products directly, as long as the total volume of unpurified PCR products in the Assembly reaction is 20% or less. If greater amounts of PCR products are used, we recommend the use of a column-based cleanup kit (Monarch PCR & DNA Cleanup Kit, NEB #T1030). In the event that multiple fragments are amplified or the presence of primer dimers is observed, we recommend optimizing the PCR step for best results. 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 and final assembled product also carry the restriction site that was used to linearize the vector it is necessary to heat inactivate the restriction enzyme or purify the cut vector if heat inactivation is not possible. 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 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 NEBuilder HiFi DNA Assembly Master Mix/Cloning Kit. 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. 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: 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 requirements for 2–3 fragment assemblies versus 4+? 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 15–20 nt overlaps. The 1 hour assembly protocol is recommended for the assembly of 4+ fragments, flanked by 20–30 nt overlaps. Q: Can I use PCR product amplified from Taq DNA polymerase? A: Yes. The additional A base at the 3´ end of PCR product will be removed during DNA assembly if it becomes a mismatched residue once fragments anneal. 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: Are there any differences between NEBuilder HIFi DNA Assembly Master Mix, the NEBuilder HiFi DNA Assembly Cloning Kit and NEBuilder HiFi DNA Assembly Bundle for Large Fragments? A: The NEBuilder HiFi DNA Assembly Master Mix in all products is the same. NEBuilder HiFi DNA Assembly Cloning Kit includes additional NEB 5-alpha chemically competent E. coli. NEBuilder HiFi DNA Assembly Bundle for Large Fragments includes 2 packs of NEBuilder HiFi DNA Assembly Master Mix (E2621S) and one box of NEB 10-Beta Competent E.Coli (20 x 50ul). Q: What type of competent cells are suitable for transformation of DNA constructs created using NEBuilder HiFi DNA Assembly Master Mix? 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 15 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. NEBuilder HiFi DNA Assemble Master Mix and NEB 5-alpha Competent E. coli are available together as the NEBuilder HiFi DNA Assembly Cloning Kit (NEB #E5520). NEBuilder HiFi and NEB 10-beta Competent E. coli are available together as the NEBuilder HiFi DNA Assembly Bundle for Large Fragments (NEB #E2623). Q: Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e., CACCACCACCACCAC)? A: 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. Alternatively, intersperse CAC and CAT his codons to interrupt this repetitive sequence. You should avoid repeating sequences at the end of an overlap. Q: Is this method applicable to the assembly of repetitive sequences? A: 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. Alternatively, consider Golden Gate Assembly for assemblies containing repetitive sequences. Q: What is the difference between NEBuilder HiFi DNA Assembly Master Mix/DNA Assembly Cloning kit/Bundle for Large Fragments kit and the current NEB Gibson Assembly Master Mix/Cloning Kit? A: The NEBuilder HiFi DNA Assembly Master Mix utilizes a high-fidelity polymerase with higher accuracy. While protocols for these kits are similar, the assembled products from NEBuilder HiFi DNA Assembly Master Mix and NEBuilder HiFi Cloning Kit will typically result in more colonies. When large DNA (> 10 kb) or multiple fragments (4+) need to be assembled, increasing the overlap region to 30 bp improves the efficiency of assembly and transformation. There are also no licensing fee requirements from NEB with the NEBuilder products. Q: Can NEBuilder® HiFi DNA Assembly master mix remove both 3′ and 5′ end mismatches? A: Yes, NEB HiFi can remove these mismatches up to 10bp from the end, allowing for the DNA ends to anneal without the overhang Q: How should fragments be prepared for assembly using NEBuilder HiFi? A: Fragments can be prepared by the following methods: PCR-generated fragments can be cleaned-up by using Monarch PCR column or Exo-CIP Rapid PCR Cleanup Kit if amplicon purity is greater than 95%. If plasmid DNA was used as template during PCR, it can be removed by DpnI treatment if necessary. If multi-bands are observed, we recommend optimizing the PCR. If this is not possible gel purification is recommended. Gel extraction can introduce guanidine thiocynate (from the dissolving buffer) that can reduce the efficiency of the assembly reaction. To minimize this contamination, trim the gel slice so that a smaller amount of gel dissolving buffer can be used. Restriction enzyme digestion of a plasmid can be performed followed by heat-inactivation or column purification. Commercially ordered fragments can be re-suspended in nuclease-free water or TE buffer and directly used in the assembly reaction. Q: I would like to miniaturize DNA assembly with NEBuilder HiFi DNA Assembly Master Mix using the Labcyte Echo Liquid Handler. Do you have any information that I can refer to help me automate DNA assembly? A: The following references by Labcyte are available: Application note: Miniaturized Multi-piece DNA Assembly Technical note: Modular DNA Assembly of PIK3CA using Acoustic Liquid Transfer in Nanoliter Volumes Technical note: Nanoliter Scale DNA Assembly Utilizing the NEBuilder HiFi Cloning Kit Q: How does overlap length affect NEBuilder HiFi DNA Assembly efficiency? A: Longer overlaps (20-30 base pairs) increase assembly efficiency. Four fragments (~20 fmol) with 15 or 20 bp overlap were assembled using NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) at 50°C for 60 minutes to create a pUC19 vector. 2µl of assembled mix was transformed into NEB 5-alpha Competent E. coli (NEB #C2987) and spread on an LB/Amp plate containing IPTG and X-gal. Blue colonies that indicated correct assembly were counted. NEBuilder HiFi DNA Assembly Mix is more efficient using a 20 bp overlap compared to a 15 bp overlap. 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.