Nebuilder hifi assembly master mix

Manufactured by New England Biolabs

Sourced in United States

NEBuilder HiFi Assembly Master Mix is a high-fidelity DNA assembly solution designed for the seamless assembly of multiple DNA fragments. The master mix contains a high-fidelity DNA polymerase and optimized buffer components to facilitate efficient and accurate DNA assembly.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

NEBuilder HiFi DNA Assembly Master Mix (2×) NEBuilder high-fidelity (HiFi) DNA assembly master mix NEBuilder Hifi DNA Assembly Master Mix (pFLAG-INTS15s-FLAG) NEBuilder HiFi DNA Assembly Master Mix NEBuilder HiFi DNA Assembly Master Mix kit NEBuilder HiFi Master Mix HiFi Assembly Master Mix NEBuilder HiFi assembly NEBuilder HiFi mix NEBuilder HiFi

8 protocols using nebuilder hifi assembly master mix

Production of DMOS DNA Registers

Cited 1 time

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

The DMOS registers are made up of DNA sequences that have at least two dTdCdR sites (R = Purine, A or G). These sites have been proven to be effective binding sites based on the research conducted by Chari^{24 (link)} and synthesized by TWIST Bioscience. To facilitate the mass production of DMOS registers, we assembled the registers into the pBR322 plasmid (New England Biolabs) using the NEBuilder© HiFi Assembly Master Mix. We inserted the register into the plasmid by cleaving the pBR322 plasmid using FastDigest restriction enzymes Bsu15I and EcoRI.
Next, we transformed the modified plasmid into NEB 5-ɑ competent Escherichia coli bacteria and grew them under Carbenicillin antibiotic resistance. To extract the assembled plasmid, we used the Monarch Plasmid Miniprep Kit (NEB).

Sadremomtaz A., Glass R.F., Guerrero J.E., LaJeunesse D.R., Josephs E.A, & Zadegan R. (2023). Digital data storage on DNA tape using CRISPR base editors. Nature Communications, 14, 6472.

+ Open protocol

+ Expand

Construction of Salmonella Mutant Strains

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

Plasmids and primers used in this study are listed in the Key Resources Table and Table S4, respectively. Primers were purchased from Integrated DNA Technologies (IDT, Skokie, IL). Mutant alleles of S. enterica serovars were constructed using λ-Red recombination as described (Datsenko and Wanner, 2000 (link); Karlinsey 2007 (link)) with primers sets listed in Table S4. All plasmids were constructed using NEBuilder® HiFi Assembly Master mix (NEB, Ipswich, MA) with the following vectors and PCR products using primer sets listed in Table S4 as follows: pJK741, pJK745, pJK747, pJK749 and pJK750 with pMPMA3Δnull-gfp digested with EcoRI and PCR products generated with gDNA from S. Typhi Ty2; pJK744, pJK746, pJK748 and pJK754 with pMPMA3Δnull-gfp and PCR products generated with gDNA from S. Typhimurium 14028s; pJK753 with pWSK130 digested with BamHI and PCR products generated with pDNA from pFPVmCherry. All mutant strains and plasmid constructs were confirmed by DNA sequencing (Genewiz, South Plainfield, NJ).

Karlinsey J.E., Stepien T.A., Mayho M., Singletary L.A., Bingham-Ramos L.K., Brehm M.A., Greiner D.L., Shultz L.D., Gallagher L.A., Bawn M., Kingsley R.A., Libby S.J, & Fang F.C. (2019). Genome-Wide Analysis of Salmonella enterica serovar Typhi in Humanized Mice Reveals Key Virulence Features. Cell host & microbe, 26(3), 426-434.e6.

+ Open protocol

+ Expand

Insertion of GFP gene into plant virus

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

Green fluorescent protein (GFP) gene sequence [60 (link)] was inserted using NEBuilder HiFi Assembly Master Mix (New England Biolabs Inc., USA) in-frame between pWX6 NIb and CP coding sequences with a duplicated cleavage sites inserted between nt 8386/8387: 15 nt (5′-CagGCcGGcGAgacc-3′, lower case indicating nucleotides changed to alter codons while retaining translated sequence) encoding amino acid sequence Q/AGET, cleaved by NIa-Pro at the beginning of the GFP gene; and 24 nt (5′-GAgGTtATcGAcGTgAAgCAcCAA-3′) encoding NIb cleavage site amino acid sequence of EVIDVKHQ/) at the end of GFP. Primers WX123/WX124 and WX126/WX127 were used to amplify the full-length GFP sequence, primers WX125/LRS764 and WX128/LRS765 for MDMV OH5 sequence, and LRS766/LRS769 were used to amplify the pJL89 vector (Additional file 1: Table S1). The GFP-encoding gene fragment was assembled into pWX6 using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs, USA), and subsequent clones were tested for infectivity.

Xie W., Marty D.M., Xu J., Khatri N., Willie K., Moraes W.B, & Stewart L.R. (2021). Simultaneous gene expression and multi-gene silencing in Zea mays using maize dwarf mosaic virus. BMC Plant Biology, 21, 208.

+ Open protocol

+ Expand

Plasmid Construction and Transformation

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

Q5^® High-Fidelity 2X Master Mix (New England Biolabs) was used for all polymerase chain reactions, followed by DpnI (New England Biolabs) digestion to remove the cell-derived plasmid template when necessary. NEBuilder HiFi Assembly Master Mix (New England Biolabs) was used for plasmid construction, followed by transformation into chemically competent NEB 5-α F’I^qE. coli (New England Biolabs), or into CopyCutter™ EPI400™ electrocompetent E. coli to increase plasmid yield, all according to manufacturer’s instructions. Transformants were selected on Lysogeny Broth (LB) agar (Lennox) plates supplemented with 50 μg mL⁻¹ kanamycin (Sigma-Aldrich) and grown overnight at 37 °C. Correct constructs were confirmed by Sanger sequencing (GENEWIZ, Inc.). Some plasmids were synthesized (Twist Bioscience). Detailed plasmid construction information is described in Supplementary Data 2. Sequences for synthesized plasmids are in Supplementary Data 3. Oligos used in plasmid construction are listed in Supplementary Data 4.

Ling C., Peabody G.L., Salvachúa D., Kim Y.M., Kneucker C.M., Calvey C.H., Monninger M.A., Munoz N.M., Poirier B.C., Ramirez K.J., St. John P.C., Woodworth S.P., Magnuson J.K., Burnum-Johnson K.E., Guss A.M., Johnson C.W, & Beckham G.T. (2022). Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering. Nature Communications, 13, 4925.

+ Open protocol

+ Expand

Maize Gene VIGS Using pWX27

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

Infectious clone pWX27 with GFP inserted between NIb and CP was used as backbone vector for a triple partial gene sequence cloning between P1 and HCPro. Three maize genes, magnesium chelatase (ZmChlI, GenBank accession no. DQ084025, target region: 946–1193), lemon white 1 (ZmIspH, GenBank accession no. NM_001175829, target region: 740–988) and phytoene desaturase (ZmPDS, GenBank accession no. L39266, target region: 538–786) were selected for VIGS analysis. The triple gene fragment, 249 nt of each gene with total length of 747 nt, was synthesized (Eurofins Genomics, USA), cloned into pMINIT2.0 vector and verified by sequencing. The triple VIGS gene fragment was then amplified by PCR with primers WX251 and WX252, and the pWX27 backbone was amplified using primers WX247 and WX250, assembled using NEBuilder HiFi Assembly Master Mix (New England Biolabs Inc., USA) in-frame between P1 and HCPro to create pWX56. The triple VIGS DNA fragment contained nine additional 5′ nt (5′-GCcGAtCCt-3′) encoding amino acid sequence ADP at the beginning of the VIGS insertion, and 33 nt 3′ (5′- GAgGTAATcGAcGTgAAgCAcCAAGCcGGcGag-3′), encoding NIb cleavage site amino acid sequence of EVIDVKHQ/AGE, cleaved by NIa-Pro), between nt 838/839 of pWX27. Recovered clones were tested for infectivity and one infectious clone, pWX56, was selected for further analysis.

+ Open protocol

+ Expand

Cloning and Assembly of Genetic Constructs

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

WT sequences of gfi1b, mCherry, and runx1 +23 enhancer were separately cloned into pJET1.2 vectors using the CloneJet PCR Cloning Kit (Thermo Fisher Scientific). After transformation, the outgrown colonies were selected for DNA isolation. The presence of the insert was confirmed by restriction enzyme digestion using BglII (NEB) followed by agarose gel electrophoresis (1% and 5%). DNA fragments were then used as a polymerase chain reaction template for the Gibson cloning reaction. Fragments were amplified using overhang primers (supplemental Table 1) and purified using a DNA Clean & Concentrator kit (Zymo Research). The pUC19-iTol2 backbone was digested with BamHI-HF (NEB) overnight at 37°C, and NEBuilder HiFi Assembly MasterMix (NEB) was used for the assembly of the fragments. The correct assembly was determined by HindIII restriction enzyme digestion and polymerase chain reaction amplification of the fragments. All transformations were performed using Escherichia coli and by performing heat shock for 30 seconds at 42°C followed by recovery in SOC Outgrowth Medium (NEB) for 1 hour. All colonies were grown in lysogeny broth medium plates supplemented with carbenicillin (50 mg/mL; 1000:1 volume-to-volume ration), and DNA from individual colonies was isolated using a QIAprep Spin Miniprep Kit (Qiagen).

Koyunlar C., Gioacchino E., Vadgama D., de Looper H., Zink J., ter Borg M.N., Hoogenboezem R., Havermans M., Sanders M.A., Bindels E., Dzierzak E., Touw I.P, & de Pater E. (2023). Gata2-regulated Gfi1b expression controls endothelial programming during endothelial-to-hematopoietic transition. Blood Advances, 7(10), 2082-2093.

+ Open protocol

+ Expand

GFP Gene Cloning and Infectivity Testing

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

GFP sequence was also cloned using NEBuilder HiFi Assembly Master Mix (New England Biolabs Inc., USA) in-frame between MDMV OH5 P1 and HCPro coding sequences with inserted NIb cleavage site sequence in pWX6 (nt 838/839), adding 12 nt 5′ (5′-GCcGAtCCtacc-3′), encoding amino acid sequence ADPT 5′, and 33 nt 3′ (5′- GAgGTAATcGAcGTgAAgCAcCAAGCcGGcGag-3′), encoding amino acid sequence EVIDVKHQ/AGE, cleaved by NIa-Pro) at the end of GFP. Nested primers WX36/WX37 and WX63/WX64 were used to amplify the full-length GFP gene sequence, and primers WX247 and WX250 were used to amplify pJL89 with MDMV sequence (Additional file 1: Table S1). The GFP gene fragment was assembled into pWX6 and recovered clones were tested for infectivity.

+ Open protocol

+ Expand

Maize VIGS Vector with Triple Gene Fragment

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

Infectious clone pWX27 with GFP inserted between NIb and CP was used as backbone vector for a triple partial gene sequence cloning between P1 and HCPro. Three maize genes, magnesium chelatase (ZmChlI, GenBank accession no. DQ084025, target region: 946-1193), lemon white 1 (ZmIspH, GenBank accession no. NM_001175829, target region: 740-988) and phytoene desaturase (ZmPDS, GenBank accession no. L39266, target region: 538-786) were selected for VIGS analysis. The triple gene fragment, 249 nt of each gene with total length of 747 nt, was synthesized (Euro ns Genomics, USA), cloned into pMINIT2.0 vector and veri ed by sequencing. The triple VIGS gene fragment was then ampli ed by PCR with primers WX251 and WX252, and the pWX27 backbone was ampli ed using primers WX247 and WX250, assembled using NEBuilder HiFi Assembly Master Mix (New England Biolabs Inc., USA) in-frame between P1 and HCPro to create pWX56. The triple VIGS DNA fragment contained nine additional 5' nt (5'-GCcGAtCCt-3') encoding amino acid sequence ADP at the beginning of the VIGS insertion, and 33 nt 3' (5'-GAgGTAATcGAcGTgAAgCAcCAAGCcGGcGag-3'), encoding NIb cleavage site amino acid sequence of EVIDVKHQ/AGE, cleaved by NIa-Pro), between nt 838/839 of pWX27. Recovered clones were tested for infectivity and one infectious clone, pWX56, was selected for further analysis.

Xie W., Marty D., Xu J., Khatri N., Willie K., Moraes W.B., & Stewart L.R. (2020). Simultaneous Gene Expression and Multi-Gene Silencing in Zea Mays Using Maize Dwarf Mosaic Virus.

+ 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!