Nebuilder hifi dna assembly

Manufactured by New England Biolabs

Sourced in United States, Germany, Japan, France, United Kingdom

NEBuilder HiFi DNA Assembly is a proprietary DNA assembly method developed by New England Biolabs. It enables the seamless and efficient assembly of multiple DNA fragments in a single, isothermal reaction.

Automatically generated - may contain errors

Lab products found in correlation

235 protocols using nebuilder hifi dna assembly

Generation of IFI16 Mutant Constructs

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

IFI16 mutant plasmid constructs were originated from a pCDNA3 human IFI16-HA tagged expression construct kindly provided by Professor Andrew Bowie, Trinity College Dublin. Overlap extension PCR was used to construct a ΔPyrin domain (consisting of amino acids 87–729 of IFI16) or a ΔHIN-A domain+HIN-B domain specific mutations (consisting of amino acids 1–191 and 460–729 of IFI16 and the point mutations K572A, K607A, R611A, S614A, K618A, N654A, K676A, K678A, K703A). Each PCR product was then recloned into a BamHI –and XhoI-digested pCCL-PGK-eGFP (ref. 52 ) together with a PCR-amplified IRES-BFP fragment by NEBuilder HiFi DNA Assembly according to manufactures instructions. For illustration see Fig. 6h.
The mBanana-cGAS fusion construct was engineered by PCR amplification of mBanana and cGAS and subsequent cloning into a NotI-digested pT2/CMV-eGFP.SV40-neo53 (link) by NEBuilder HiFi DNA Assembly according to manufactures instructions.

Jønsson K.L., Laustsen A., Krapp C., Skipper K.A., Thavachelvam K., Hotter D., Egedal J.H., Kjolby M., Mohammadi P., Prabakaran T., Sørensen L.K., Sun C., Jensen S.B., Holm C.K., Lebbink R.J., Johannsen M., Nyegaard M., Mikkelsen J.G., Kirchhoff F., Paludan S.R, & Jakobsen M.R. (2017). IFI16 is required for DNA sensing in human macrophages by promoting production and function of cGAMP. Nature Communications, 8, 14391.

+ Open protocol

+ Expand

Generation of Plasmids for NELF Manipulation

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Three plasmids were generated for this study: (1) Cas9 vector to target the C terminus of Nelfb gene: PX459 vector (Addgene 62988) was digested using BbsI-HF (NEB) and single guide RNA targeting Nelfb was annealed (Ran et al. 2013 (link)). (2) Homology-directed repair (HDR) vector containing the insert FKBP^F36V tag, 2× HA tag, self-cleaving P2A sequence, and puromycin resistance, flanked by 1-kb Nelfb HDR sequences: The insert was obtained from pCRIS-PITCHv2-dTAG-BSD (Addgene 91795) (Nabet et al. 2018 (link)). The plasmid backbone (pBluescript), Nelfb HDR sequences, and the insert were amplified using Q5 polymerase (NEB), and the plasmid was constructed using NEBuilder HiFi DNA assembly (NEB). (3) Nelfe-EGFP vector as a fluorescent reporter of NELF bodies: Nelfe cDNA was amplified using Q5 polymerase (NEB). Linker-EGFP and PGK backbone were amplified from pHaloTag-EGFP (Addgene 86629) and PGKneobpa (Addgene 13442), respectively. The plasmid was constructed using NEBuilder HiFi DNA assembly (NEB).

Abuhashem A., Chivu A.G., Zhao Y., Rice E.J., Siepel A., Danko C.G, & Hadjantonakis A.K. (2022). RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription. Genes & Development, 36(13-14), 770-789.

+ Open protocol

+ Expand

Multifunctional Lentiviral Plasmid Construction

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

The pcDNA5 GASTM-3-RVG-10-Lamp2b-HA plasmid was obtained from Addgene (#71295) and the GASTM-RVG cDNA sequence was replaced by the glycosylation sequence GNSTM followed by BbsI restriction sites using PCR and subsequent NEBuilder HiFi DNA assembly (New England Biolabs) according to manufacturer’s instructions. Afterward, the open-reading frame containing an N-terminal signal peptide (SP) was cloned into the pJET1.2/blunt cloning vector (Thermo Scientific) by PCR and NEBuilder HiFi DNA assembly, after which specific targeting peptide (TP) tags flanked by BbsI restriction sites were incorporated into the BbsI cut by T4 DNA ligase (New England Biolabs) according to manufacturer’s instructions. Complete SP-GNSTM-3-TP-10-Lamp2b-HA cDNA sequences were amplified by PCR to contain BamHI and NotI overhangs and cloned into the pHAGE2-EF1alpha-IRES-PuroR-WPRE lentiviral vector (Wilson et al., 2008 (link); de Jong et al., 2020 (link)). TP tags included: TP-FLAG (DYKDDDDK), TP1 (RGD-4C; ACDCRGDCFCG), TP2 (CRPPR) and TP-9R (RRRRRRRRR). 3; 10: glycine-serine amino acid spacers. Oligo sequences are provided in Supplementary Table S1. Primer sequences are available upon request.

Roefs M.T., Heusermann W., Brans M.A., Snijders Blok C., Lei Z., Vader P, & Sluijter J.P. (2022). Evaluation and manipulation of tissue and cellular distribution of cardiac progenitor cell-derived extracellular vesicles. Frontiers in Pharmacology, 13, 1052091.

+ Open protocol

+ Expand

Cloning of AAV-based optoGenetic Vectors

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

To obtain pAAV-hSyn-GFP-IRES-tTA vector, DNA fragment of GFP-IRES-tTA from pAAV-TRE-fDIO-GFP-IRES-tTA^{66 (link)} was inserted into the pAAV-hSyn-DIO-hM3D(Gq)-mCherry^{67 (link)} lacking DIO-hM3D(Gq)-mCherry region by SalI and EcoRV digestion using the NEBuilder HiFi DNA assembly (NEB). DNA fragment of the hSyn promoter regions was exchanged for the L7 minimal promoter^{68 (link)} to construct pAAV-L7-GFP-IRES-tTA.
To obtain AAV-TRE-HA-mGlu1(WT) or AAV-TRE-HA-mGlu1(N264H), DNA fragment of TRE promoter and WPRE3^{35 (link)} were obtained from pAAV-TRE-fDIO-GFP-IRES-tTA^{66 (link)}, and SV40 late polyA was from pCI-neo vector (Promega). DNA fragment of HA-tagged mGlu1 was obtained by inserting HA-tag after the membrane localization signal of mGlu1. These DNA fragments were inserted into pAAV-hSyn-DIO-hM3D(Gq)-mCherry^{67 (link)} lacking DNA fragment from hSyn promoter to hGH polyA by MluI and PmlI digestion using the NEBuilder HiFi DNA assembly (NEB). See Supplementary Fig. 14a for illustration of these vector constructs. pAAV-hSyn-DIO-hM3D(Gq)-mCherry and pAAV-TRE-fDIO-GFP-IRES-tTA are gifts from Bryan Roth (Addgene plasmid #44361) and Minmin Luo (Addgene plasmid #118026), respectively.

Ojima K., Kakegawa W., Yamasaki T., Miura Y., Itoh M., Michibata Y., Kubota R., Doura T., Miura E., Nonaka H., Mizuno S., Takahashi S., Yuzaki M., Hamachi I, & Kiyonaka S. (2022). Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue. Nature Communications, 13, 3167.

+ Open protocol

+ Expand

Constructing Thermometer-Controlled Bacterial Gene Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

The initial plasmid backbone was synthesized from VectorBuilder (VectorBuilder Inc, Chicago, IL, USA). The 5′-UTR of thermometer candidates were placed directly upstream of a heat-stable galactosidase from Bacillus stearothermophilus and driven by a pBAD promoter (pBAD: galactosidase). ATG (start codon) in the thermometer sequences replaces the first ATG of bgaB.
NEBuilder® HiFi DNA Assembly was used to insert sequences into the same plasmid backbone described above. NEBuilder® HiFi DNA Assembly was performed according to the manufacturer's protocol. A previously described vector used to validate the blyA thermometer (Tong et al. 2023 ) (VectorBuilder ID: VB220225-1020jdm, can be retrieved from https://en.vectorbuilder.com/design/retrieve.html) was used as the backbone for plasmid construction. NEBuilder Assembly Tool 2.0 was used to design fragments. Sequences of mutants for -galactosidase assay (Table S2) were designed with the following complementary flanking sequences to the VB220225-1020jdm plasmid. 5′-ATACCCGTTTTTTGGGCTAA -Sequences for -galactosidase assay (Table S2) -

Sharts D.M., Almanza M.T., Banks A.V., Castellanos A.M., Hernandez C.G.O., Lopez M.L., Rodriguez D., Tong A.Y., Segeberg M.R., Passalacqua L.F.M, & Abdelsayed M.M. (2024). Robo-Therm, a pipeline to RNA thermometer discovery and validation. RNA (New York, N.Y.), 30(7).

+ Open protocol

+ Expand

Engineered Plasmids for Frameshifting Analysis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

The p2FL constructs were generated based on the reported SF reporter plasmid.²⁶ (link) The original influenza polymerase basic 2 (PB2) was replaced by eGFP (abbreviated as GFP) and mDsRed (abbreviated as RFP) (a gift from Dr. Hua-Lin Wu, National Cheng Kung University). In this newly constructed p2FL plasmid, GFP would be produced in 0 frame and RFP in −1 frame. The slippery sequence, spacer (3, 5, 7, 9, 11 nts), and targeting sequences (v1, v2, v3, v4, SARS CoV2 PK) were cloned between GFP and RFP of p2FL using standard cloning or NEBuilder HiFi DNA Assembly (NEB). The p2Luc-SARS CoV2 PK plasmid was created by inserting SARS-CoV-2 frameshifting context into p2Luc⁶⁸ (link) (a gift from Dr. Atkins, University College Cork) through NEBuilder HiFi DNA Assembly (NEB). The pET-21a_LbCas12a-3HA-6His was generated by PCR-mediated deletion from Addgene plasmids pET-21a_LbCas12a-2xNLS-3HA-6His⁵⁰ (link) (#114366). The constructs are confirmed by Sanger sequencing (Genomics, Taiwan). Please see Table S3 for the detail oligonucleotide sequences.

Huang S.H., Chen S.C., Wu T.Y., Chen C.Y, & Yu C.H. (2023). Programmable modulation of ribosomal frameshifting by mRNA targeting CRISPR-Cas12a system. iScience, 26(12), 108492.

+ Open protocol

+ Expand

Lentiviral Dual Reporter Construction

Check if the same lab product or an alternative is used in the 5 most similar protocols

mCherry and P2A-eGFP were PCR amplified with primers containing a BstBI restriction site and adequate overhangs (primer sequences provided in Table S2). Both PCR fragments were inserted into XbaI and EcoRI digested lentiviral backbone (Addgene #52962) in a single NEBuilder HiFi DNA Assembly (NEB) reaction. The resulting vector was digested with BstBI. Annealed SARS-CoV-2 or HIV FSE (PRF-1 and PRF0) oligonucleotides were inserted by NEBuilder HiFi DNA Assembly (NEB). The resulting vector (Lenti-mCh-PRF-P2A-eGFP) was then used to generate lentivirus. 1x10⁶ HEK293T cells were seeded per well in a 6-well plate. 24 hours after seeding, cells were transfected with 1 μg Lenti-mCh-PRF-P2A-eGFP, 0.6 μg psPAX2 (Addgene #12260), and 0.4 μg pMD2.G (Addgene #12259) using 5 μl FugeneHD (Promega) transfection reagent. The medium was changed 24 hours post transfection and 48 hours after transfection, viral supernatant was collected and passed through a 0.45 μm filter. HCT116 cells were transduced with viral supernatant in the presence of 5 μg/ml polybrene at a low multiplicity of infection. 48 hours after transduction, single mCherry and eGFP positive cells were sorted into 96-well plates and clonal cell lines were established.

Rehfeld F., Eitson J.L., Ohlson M.B., Chang T.C., Schoggins J.W, & Mendell J.T. (2023). CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication. Cell Reports, 42(2), 112076.

+ Open protocol

+ Expand

SARS-CoV-2 Spike Mutation Introduction

Check if the same lab product or an alternative is used in the 5 most similar protocols

The introduction of the B.1.525 lineage mutations into pCAGS-Spike Sars-CoV2 plasmids (BEI) was performed by restriction digest of the plasmid, followed by NEBuilder HiFi DNA assembly (New England Biolabs) with either amplified by PCR products or synthesized DNA fragments (Genewiz). The full modified sequences were verified and confirmed by Sanger sequencing (Supplementary Methods).

Ozer E.A., Simons L.M., Adewumi O.M., Fowotade A.A., Omoruyi E.C., Adeniji J.A., Olayinka O.A., Dean T.J., Zayas J., Bhimalli P.P., Ash M.K., Maiga A.I., Somboro A.M., Maiga M., Godzik A., Schneider J.R., Mamede J.I., Taiwo B.O., Hultquist J.F, & Lorenzo-Redondo R. (2022). Multiple expansions of globally uncommon SARS-CoV-2 lineages in Nigeria. Nature Communications, 13, 688.

+ Open protocol

+ Expand

Venus Expression Construct Generation

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Venus expression constructs were designed in SnapGene (v.4.3.11) and generated by integrating PCR-amplified (Q5 Hot Start High Fidelity, M0515, New England Biolabs) DNA fragments into plasmid pODC53 (Caspari, 2020 (link)) upstream of Venus using Gibson assembly (NEBuilder HiFi DNA assembly, E5520S, New England Biolabs). Chlamydomonas TP sequences were amplified from genomic DNA extracted from strain T222+ (CC-5101). Templates for codon-optimized HA-RAMP, RP, and R→K modified TP sequences were obtained by gene synthesis (Eurofins Genomics). Correct assembly was verified by sequencing (Eurofins Genomics). Linear transformation cassettes were generated through restriction digestion with EcoRV (New England Biolabs).

Caspari O.D., Garrido C., Law C.O., Choquet Y., Wollman F.A, & Lafontaine I. (2023). Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts. Plant Communications, 4(4), 100555.

+ Open protocol

+ Expand

Domestication of EV-A71 Molecular Clone

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Because SPINE and DIMPLE use BsaI and BsmBI type IIS restriction enzymes to assemble mutant libraries, we first ‘domesticated’ the original EV-A71 strain Tainan/4643/98 molecular clone (Genbank accession: AF304458.1) ^{51 (link)} by removing ten BsmBI or BsaI sites to improve efficiency of downstream assembly steps. Parts of the molecular clone which did not include BsaI and BsmBI restriction sites were generated by PCR. In parallel, fragments containing the restriction sites were ordered as gene fragments (Twist Biosciences). All the fragments (six in total) had a 30 bp overlap and were assembled by NEBuilder HiFi DNA Assembly (New England Biolabs, E5520S). We compared virus production between the original and domesticated clones and observed that viral growth in RD cells was comparable with rescued virus titers reaching around 10⁶ TCID₅₀/mL.

Bakhache W., Orr W., McCormick L, & Dolan P.T. (2024). Uncovering Structural Plasticity of Enterovirus A through Deep Insertional and Deletional Scanning. Research Square.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!