Nebuilder hifi dna assembly master mix kit

Manufactured by New England Biolabs

Sourced in United States

NEBuilder HiFi DNA Assembly Master Mix kit is a high-fidelity DNA assembly solution that enables the seamless assembly of multiple DNA fragments in a single reaction. The kit provides a simple and efficient method for constructing recombinant DNA molecules.

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Lab products found in correlation

Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Hek293t, by American Type Culture Collection (1 mentions) Pcdna3, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) X tremegene 9 dna transfection reagent, by Merck Group (1 mentions) Mycoalert, by Lonza (1 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions) Neuro 2a ccl 131, by American Type Culture Collection (1 mentions) Pspax2, by Addgene (1 mentions) Pwpxl, by Addgene (1 mentions) Pmd2 g, by Addgene (1 mentions) Pierce high capacity neutravidin agarose, by Thermo Fisher Scientific (1 mentions) Hifi dna assembly master mix kit, by New England Biolabs (1 mentions) Pypq141b, by Addgene (1 mentions) Pypq266a, by Addgene (1 mentions)

Close spelling variants of this product

NEBuilder HiFi DNA Assembly Master Mix (514 citations) NEBuilder HiFi DNA Assembly (235 citations) NEBuilder HiFi DNA Assembly kit (168 citations) HiFi DNA Assembly Master Mix (94 citations) HiFi DNA Assembly (60 citations) HiFi DNA Assembly kit (47 citations) NEBuilder HiFi (29 citations) NEBuilder HiFi Assembly kit (27 citations) HiFi Assembly Master Mix (22 citations) HiFi assembly kit (21 citations)

7 protocols using nebuilder hifi dna assembly master mix kit

Recombinant Protein Expression Constructs

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Full-length cDNA of N protein was synthesized according to the SARS-CoV-2 genomic reference sequences but with alternative codons (Integrated DNA Technologies). N-FLAG was inserted into the backbone of pEGFP-C3 plasmid (with GFP removed), AcGFP-N was inserted into the backbone of pAcGFP1-C1, and His-SUMO-N was inserted into the backbone of pETite-HisSUMO (Lucigen) using NEBuilder HiFi DNA Assembly Master Mix kit (NEB; E2621). Using the same methods, human G3BP1 cDNA was inserted into pGEX-2T; G3BP1-EGFP, mCherry-nsP3–25mer (449–472) was inserted into the backbone of pEGFP-C3 plasmid (with GFP removed); EGFP-N-25mer (1–25), EGFP-caprin1–21mer (361–381), EGFP-USP10–25mer (1–25), EGFP-nsP3–25mer (449–473) were inserted into the backbone with pEGFP-C3. Truncations of N-FLAG (48–419, 175–419, 210–419, 1–361, 1–267, 1–209), GST-G3BP1 (GST-NTF2L, GST-deltaNTF2L), and AcGFP-N (1–47, 1–35, 1–25) were created using NEBuilder HiFi DNA Assembly Master Mix kit. Point mutations of N-FLAG (F17A and F17W), AcGFP-N (F17A), GFP-tagged peptides (EGFP-N-25mer-F17A, EGFP-caprin1–21mer-F372A, EGFP-USP10–25mer-F10A and EGFP-nsP3–25mer-F451/468A), GST-NTF2L (F33W), G3BP1-GFP (V11A, F112A, F33A, F33W, F15A, F15W, F124A, F124W) were created by the Q5 Site-Directed Mutagenesis kit (NEB; E0554S).

Yang Z., Johnson B.A., Meliopoulos V.A., Ju X., Zhang P., Hughes M.P., Wu J., Koreski K.P., Chang T.C., Wu G., Hixon J., Duffner J., Wong K., Lemieux R., Lokugamage K.G., Alvardo R.E., Crocquet-Valdes P.A., Walker D.H., Plante K.S., Plante J.A., Weaver S.C., Kim H.J., Meyers R., Schultz-Cherry S., Ding Q., Menachery V.D, & Taylor J.P. (2023). Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication. bioRxiv.

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Cell lines and transfection methods

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HEK293T (CRL-11268), HeLa (CCL-2), and Neuro2A (CCL-131) cells were obtained from American Type Culture Collection (ATCC) and were checked for mycoplasma contamination with the use of MycoAlert (Lonza). They were cultured under an atmosphere of 5% CO₂ at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Life Technologies) and antibiotics. For transient expression, cDNAs were subcloned into pcDNA3 (Invitrogen) with the use of a NEBuilder HiFi DNA Assembly Master Mix kit (New England BioLabs) and transfection was performed with the use of the X-tremeGENE 9 DNA Transfection Reagent (Sigma-Aldrich). For RNA interference, Neuro2A cells were transfected with 10 nM siRNAs (VDAC1, SASI_Mm02_00321250; VDAC2, SASI_Mm02_00321253; VDAC3, SASI_Mm01_00110006; or Universal Negative Control #1, SIC-001) with the use of the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific).

Mise S., Matsumoto A., Shimada K., Hosaka T., Takahashi M., Ichihara K., Shimizu H., Shiraishi C., Saito D., Suyama M., Yasuda T., Ide T., Izumi Y., Bamba T., Kimura-Someya T., Shirouzu M., Miyata H., Ikawa M, & Nakayama K.I. (2022). Kastor and Polluks polypeptides encoded by a single gene locus cooperatively regulate VDAC and spermatogenesis. Nature Communications, 13, 1071.

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Light-Inducible STING Activation Constructs

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The plasmids pDONR223-TBK1-WT (#82285), pTRIP-GFP-IRF3 (#127663), STING-V1 (#124262), pMSCV-IRES-GFP-MyD88-CpLxIS (#131348), and the packing vectors pMD2.G (#12259) and psPAX2 (#12260), as well as the lentiviral vector pWPXL (#12257, Addgene), were obtained from Addgene (Watertown, MA, USA). To generate light-inducible constructs to recapitulate STING activation, two copies of cDNA encoding the C-terminal tail of human STING (STING-CTT) and/or the mouse STING were inserted into the mCherry-CRY2PHR or mCherry-CRY2clust vectors by using the NEBuilder HiFi DNA Assembly Master Mix kit (NEB, (Ipswich, MA, USA). TBK1-YFP was made by inserting the cDNA encoding human TBK1 (#82285, Addgene) into the pEYFP-N1 vector using the KpnI and AgeI restriction sites. To generate stable cell lines, constructs containing both mCherry-CRY2PHR-pLxIS and mCherry-CRY2clust-pLxIS, along with their respective controls (mCherry-CRY2PHR and mCherry-CRY2clust), were inserted into pWPXL between the BamHI and EcoRI sites. All constructs were validated through Sanger DNA sequencing. In a further modified version, the pMSCV-GFP-IRES-CRY2clust-pLxIS plasmid, along with the empty vector pMSCV-GFP-IRES-CRY2clust-stop as control, was used for retroviral transduction of isolated mouse dendritic cells.

Dou Y., Chen R., Liu S., Lee Y.T., Jing J., Liu X., Ke Y., Wang R., Zhou Y, & Huang Y. (2023). Optogenetic engineering of STING signaling allows remote immunomodulation to enhance cancer immunotherapy. Nature Communications, 14, 5461.

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Cloning Human eIF3 Subunits

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Complementary DNAs for human eIF3 subunits containing the V5 tag were amplified by the polymerase chain reaction (PCR) and subcloned into the EcoRV site of pcDNA3 (Invitrogen) with the use of a NEBuilder HiFi DNA Assembly Master Mix kit (New England Biolabs).

Ichihara K., Matsumoto A., Nishida H., Kito Y., Shimizu H., Shichino Y., Iwasaki S., Imami K., Ishihama Y, & Nakayama K.I. (2021). Combinatorial analysis of translation dynamics reveals eIF2 dependence of translation initiation at near-cognate codons. Nucleic Acids Research, 49(13), 7298-7317.

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Proximity Proteomics of FMRP-miniTurbo

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MiniTurbo was cloned from C1(1-29)-miniTurbo-V5_pCDNA3 (#107174, Addgene) and fused to the C-terminus of FMRP to form FMRP-miniTurbo using a NEBuilder^® HiFi DNA Assembly Master Mix kit (E2621S, NEB). Three 10 cm dishes containing 70% confluent HEK293T cells were transfected with FMRP-miniTurbo using PEI (1 μg/ml) for 24 h, 500 μM biotin was added to cells for 6 h, and cells were collected and lysed in 1 ml of lysis buffer. After centrifugation at 14,000 × g for 15 min, supernatant was collected and incubated with 25 μl Pierce^™ High Capacity NeutrAvidin^™ Agarose (29202, Thermofisher) at 4°C overnight. Beads were collected by centrifuging at 500 × g for 5 min and washed twice with lysis buffer. Finally, beads were resuspended in loading buffer and boiled for 10 min followed by Western blot analysis. The boiled samples were loaded on 10% SDS-PAGE gel and enriched on the top of the gel after running at short time. The gel was performed Coomassie Brilliant Blue Stain, and the enriched band was cut to do HPLC-MS in Stanford University Mass Spectrometry (SUMS) to identify the proximity proteins.

Geng J., Khaket T.P., Pan J., Li W., Zhang Y., Ping Y., Sillero M.I, & Lu B. (2023). Deregulation of ER-Mitochondria Contact Formation and Mitochondrial Calcium Homeostasis Mediated by VDAC in Fragile X Syndrome. Developmental cell, 58(7), 597-615.e10.

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Recombinant Expression of SARS-CoV-2 N Protein

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Full-length cDNA of the N protein was synthesized using the SARS-CoV-2 genomic reference sequences, but with alternative codons (Integrated DNA Technologies). N-FLAG was inserted into the backbone of pEGFP-C3 plasmid (with GFP removed). AcGFP-N was inserted into the backbone of pAcGFP1-C1 plasmid. His-SUMO-N was inserted into the backbone of pETite-HisSUMO plasmid (Lucigen) using NEBuilder HiFi DNA Assembly Master Mix kit (NEB; E2621). Similarly, human G3BP1 cDNA was inserted into the pGEX-2T plasmid. G3BP1-EGFP, mCherry-nsP3–25mer (449–472) were inserted into the backbone of pEGFP-C3 plasmid (with GFP removed); EGFP-N-25mer (1–25), EGFP-caprin1–21mer (361–381), EGFP-USP10–25mer (1–25), EGFP-nsP3–25mer (449–473) were inserted into the backbone of pEGFP-C3 plasmids. Truncations of N-FLAG (48–419, 175–419, 210–419, 1–361, 1–267, 1–209), GST-G3BP1 (GST-NTF2L, GST-deltaNTF2L), and AcGFP-N (1–47, 1–35, 1–25) were created using the NEBuilder HiFi DNA Assembly Master Mix kit. Point mutations of N-FLAG (F17A and F17W), AcGFP-N (F17A), GFP-tagged peptides (EGFP-N-25mer-F17A, EGFP-caprin1–21mer-F372A, EGFP-USP10–25mer-F10A and EGFP-nsP3–25mer-F451/468A), GST-NTF2L (F33W), G3BP1-GFP (V11A, F112A, F33A, F33W, F15A, F15W, F124A, F124W) were created using the Q5 Site-Directed Mutagenesis kit (NEB; E0554S).

Yang Z., Johnson B.A., Meliopoulos V.A., Ju X., Zhang P., Hughes M.P., Wu J., Koreski K.P., Clary J.E., Chang T.C., Wu G., Hixon J., Duffner J., Wong K., Lemieux R., Lokugamage K.G., Alvarado R.E., Crocquet-Valdes P.A., Walker D.H., Plante K.S., Plante J.A., Weaver S.C., Kim H.J., Meyers R., Schultz-Cherry S., Ding Q., Menachery V.D, & Taylor J.P. (2024). Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity. Cell reports, 43(3), 113965.

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CRISPR-Based Genome and Base Editing in Rice and Tomato

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For genome editing in rice, the target sites (Table S3) were generated through the online CRISPR‐BETS software. The corresponding oligos (Table S4) were synthesized, annealed and ligated into pGEL031 (Addgene #137900) and pGEL035 (Addgene #137903; Tang et al., 2018b ), for targeted mutagenesis by Cas9 and C‐to‐T base editing by nCas9(D10A)‐PmCDA1‐UGI respectively. The resulting T‐DNA expression vectors were summarized in Table S5. The CBE vector systems used in this study were summarized in Table S6.
For base editing in tomato, the target sites (Table S3) were generated by CRISPR‐BETS. Similarly, paired oligos (Table S4) for each target site were generated, annealed and inserted into pYPQ141B (Addgene #69291; Lowder et al., 2015 (link)). The nCas9(D10A)‐NG‐PmCDA1 vector, pYPQ266A (Addgene # 173176), was prepared using NEBuilder® HiFi DNA Assembly Master Mix kit (NEB, catalog #E2621*) from PCR‐amplified fragments of vectors pYPQ266 (Ren et al., 2021a (link)) and a zCas9‐NG fragment. The T‐DNA vectors were generated in three‐way Gateway reactions with one attL1‐attR5 base editor entry clone such as pYPQ266 (Addgene #164713), pYPQ266E (Addgene #161521) or pYPQ266A (Addgene # 173176), one attL5‐attL2 pYPQ141B vector containing one cloned gRNA and the attR1‐attR2 destination vector pCGS710, according to our previously established protocol (Sretenovic et al., 2021b (link)).

Wu Y., He Y., Sretenovic S., Liu S., Cheng Y., Han Y., Liu G., Bao Y., Fang Q., Zheng X., Zhou J., Qi Y., Zhang Y, & Zhang T. (2021). CRISPR‐BETS: a base‐editing design tool for generating stop codons. Plant Biotechnology Journal, 20(3), 499-510.

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