one step multi fragment gibson assembly reaction (New England Biolabs)

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New England Biolabs one step multi fragment gibson assembly reaction
One Step Multi Fragment Gibson Assembly Reaction, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/gibson+assembly+reaction/Gibson+Assembly+Master+Mix/pmc13618761-313-6-11
Average 99 stars, based on 6301 article reviews

one step multi fragment gibson assembly reaction - by Bioz Stars, 2026-10

99/100 stars

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Clone Assay:

Article Title: Membrane-associated periodic skeleton regulates major forms of endocytosis in neurons through a signaling-driven positive feedback loop
Article Snippet: CF583R-conjugated secondary antibodies were made by conjugating the unlabeled secondary antibodies with CF583R succinimidyl ester (Biotium, 96084) to achieve a labeling efficiency of ∼2 dyes per antibody, as previously described( ). .. Plasmids and gene segments used in this study were purchased from Addgene, GeneCopoeia, and Twist Bioscience, and subsequently cloned into the lentiviral expression vector FUGW (Addgene, Plasmid #14883), through Gibson assembly reaction (New England Biolabs, E5510S) according to the manufacturer’s instructions. .. Coding sequences for CAV1, FLOT1, SH3GL1 (synthesized by Twist Bioscience), APP695 (Addgene, Plasmid #114193), and Basp1 (GenoCopoeia, Plasmid # EX-Mm10271-M98) were fused to a C-terminal GFP via a flexible polypeptide linker and cloned into the FUGW vector, generating GFP-tagged constructs for caveolin-1, flotillin-1, endophilin-A2, APPwt and Basp1.

Article Title: Membrane-associated periodic skeleton regulates major forms of endocytosis in neurons through a signaling-driven positive feedback loop.
Article Snippet: The following secondary antibodies were used in this study: Alexa Fluor 647–conjugated donkey anti–guinea pig immunoglobulin G (IgG) antibody 1:800 for IF (Jackson ImmunoResearch, 706-605-148), Alexa Fluor 647–conjugated donkey anti–rabbit IgG antibody 1:800 for IF (Jackson ImmunoResearch, 711- 605-152), Alexa Fluor 647– conjugated donkey anti–mouse IgG antibody 1:800 for IF (Jackson ImmunoResearch, 715- 605- 151), Alexa Fluor 647–conjugated donkey anti–rat IgG antibody 1:800 for IF (Jackson ImmunoResearch, 712-165-153), Alexa Fluor 647–conjugated donkey anti–goat IgG antibody 1:800 for IF (Jackson ImmunoResearch, 705- 605- 147), Cy3conjugated donkey anti–rabbit IgG antibody 1:800 for IF (Jackson ImmunoResearch, 711- 165- 152), Cy3- conjugated donkey anti– mouse IgG antibody 1:800 for IF (Jackson ImmunoResearch, 715- 165-151), Cy3- conjugated donkey anti–guinea pig IgG antibody 1:800 for IF (Jackson ImmunoResearch, 706- 165-148), Cy3- conjugated donkey anti–rat IgG antibody 1:800 for IF (Jackson ImmunoResearch, 712- 165-153), Alexa Fluor 488–conjugated donkey anti–mouse IgG antibody 1:800 for IF (Jackson ImmunoResearch, 715- 545-150), Alexa Fluor 488–conjugated donkey anti–guinea pig IgG antibody 1:800 for IF (Jackson ImmunoResearch, 706- 545-148), Alexa Fluor 488– conjugated donkey anti–rabbit IgG antibody 1:800 for IF (Jackson ImmunoResearch, 711- 545- 152), donkey anti–mouse IgG antibody (Jackson ImmunoResearch, 715- 005- 151), donkey anti–rat IgG antibody (Jackson ImmunoResearch, 712- 005- 153), donkey anti–rabbit IgG antibody (Jackson ImmunoResearch, 711- 005- 152), and donkey anti–chicken IgG antibody (Jackson ImmunoResearch, 703- 005- 155) CF583R- conjugated secondary antibodies were made by conjugating the unlabeled secondary antibodies with CF583R succinimidyl ester (Biotium, 96084) to achieve a labeling efficiency of ~2 dyes per antibody, as previously described (58). .. Lentivirus production and transduction of hippocampal neurons Plasmids and gene segments used in this study were purchased from Addgene, GeneCopoeia, and Twist Bioscience and subsequently cloned into the lentiviral expression vector FUGW (Addgene, plasmid #14883), through Gibson assembly reaction (New England Biolabs, E5510S) according to the manufacturer’s instructions. .. Coding sequences for CAV1, FLOT1, SH3GL1 (synthesized by Twist Bioscience), APP695 (Addgene, plasmid #114193), and Basp1 (GenoCopoeia, plasmid # EX- Mm10271- M98) were fused to a C- terminal GFP via a flexible polypeptide linker and cloned into the FUGW vector, generating GFP- tagged constructs for Cav1, Flot1, EndoA2, APPwt, and Basp1.

Article Title: Identification of target genes and regulatory networks for bone mineral density GWAS loci through systematic targeting and inhibition
Article Snippet: .. We synthesized the BMD STING-seq gRNA library oligos and cloned them into the plasmid lentiGuideFE-Puro (Addgene 170069) immediately 3′ of the U6 promoter using the Gibson assembly reaction, following the manufacturer’s recommendations (NEB E2621S). .. We amplified the plasmid library by transfecting electrocompetent Stbl4 E. coli cells and then isolated plasmid DNA (ThermoFisher 11635018, K21007).

Expressing:

Article Title: Membrane-associated periodic skeleton regulates major forms of endocytosis in neurons through a signaling-driven positive feedback loop
Article Snippet: CF583R-conjugated secondary antibodies were made by conjugating the unlabeled secondary antibodies with CF583R succinimidyl ester (Biotium, 96084) to achieve a labeling efficiency of ∼2 dyes per antibody, as previously described( ). .. Plasmids and gene segments used in this study were purchased from Addgene, GeneCopoeia, and Twist Bioscience, and subsequently cloned into the lentiviral expression vector FUGW (Addgene, Plasmid #14883), through Gibson assembly reaction (New England Biolabs, E5510S) according to the manufacturer’s instructions. .. Coding sequences for CAV1, FLOT1, SH3GL1 (synthesized by Twist Bioscience), APP695 (Addgene, Plasmid #114193), and Basp1 (GenoCopoeia, Plasmid # EX-Mm10271-M98) were fused to a C-terminal GFP via a flexible polypeptide linker and cloned into the FUGW vector, generating GFP-tagged constructs for caveolin-1, flotillin-1, endophilin-A2, APPwt and Basp1.

Plasmid Preparation:

Article Title: Membrane-associated periodic skeleton regulates major forms of endocytosis in neurons through a signaling-driven positive feedback loop
Article Snippet: CF583R-conjugated secondary antibodies were made by conjugating the unlabeled secondary antibodies with CF583R succinimidyl ester (Biotium, 96084) to achieve a labeling efficiency of ∼2 dyes per antibody, as previously described( ). .. Plasmids and gene segments used in this study were purchased from Addgene, GeneCopoeia, and Twist Bioscience, and subsequently cloned into the lentiviral expression vector FUGW (Addgene, Plasmid #14883), through Gibson assembly reaction (New England Biolabs, E5510S) according to the manufacturer’s instructions. .. Coding sequences for CAV1, FLOT1, SH3GL1 (synthesized by Twist Bioscience), APP695 (Addgene, Plasmid #114193), and Basp1 (GenoCopoeia, Plasmid # EX-Mm10271-M98) were fused to a C-terminal GFP via a flexible polypeptide linker and cloned into the FUGW vector, generating GFP-tagged constructs for caveolin-1, flotillin-1, endophilin-A2, APPwt and Basp1.

Article Title: A near-complete map of human cytosolic degrons and their relevance for disease
Article Snippet: The DNA concentration of the extracted bands was measured by the Qubit 1× dsDNA High Sensitivity (HS) Kit (Thermo Fisher Scientific). .. The linearized p2127 vector was mixed with each of the amplified oligo sublibraries with a molecular ratio of 1:4 to perform a Gibson assembly reaction (New England Biolabs) following the manufacturer’s instructions. .. The Gibson assembly reaction was purified with the Zymo Clean and Concentrator-5 kit and eluted in 12 μl of nuclease-free water.

Amplification:

Transduction:

Purification:

Article Title: Single-cell CRISPR activation screens in primary B cells discover gene regulatory mechanisms for hundreds of autoimmune risk loci
Article Snippet: To clone the MPRA library into lentiviral vector pLS-mP (a gift from Nadav Ahituv; Addgene #81225), 125 ng of the oligonucleotide pool DNA was amplified in 4 × 50 μL reactions with 0.5 μM MPRAlib_PCR1_FW and MPRAlib_PCR1_RV primers and following conditions: 98°C for 1 min, (98°C for 10 s, 60°C for 15 s, 72°C for 20 s) × 5 cycles, 72°C for 5 min. 150 ng of purified PCR product split into 4 × 50 μL reactions and amplified with 0.5 μM MPRAlib_PCR2_FW and MPRAlib_BC_PCR2_RV primers under following conditions: 98°C for 2 min, (98°C for 15 s, 60°C for 20 s, 72°C for 30 s) × 4 cycles, 72°C for 5 min. At this step, a unique 13 bp molecular barcode (from primer MPRA_PCR2_FW) was added to each CRE. .. The purified product from the second PCR reaction (120 ng) was used in a Gibson assembly reaction with 1 μg pLS-mP linearised with Sbfi-HF (NEB) and AgeI-HF (NEB). .. The purified ligation product was transformed into Endura Electrocompetent Cells (Lucigen).

Polymerase Chain Reaction:

other:

Article Title: Membrane-associated periodic skeleton regulates major forms of endocytosis in neurons through a signaling-driven positive feedback loop
Article Snippet: The following secondary antibodies were used in this study: Alexa Fluor 647–conjugated donkey anti–guinea pig immunoglobulin G (IgG) antibody 1:800 for IF (Jackson ImmunoResearch, 706-605-148), Alexa Fluor 647–conjugated donkey anti–rabbit IgG antibody 1:800 for IF (Jackson ImmunoResearch, 711-605-152), Alexa Fluor 647–conjugated donkey anti–mouse IgG antibody 1:800 for IF (Jackson ImmunoResearch, 715-605-151), Alexa Fluor 647–conjugated donkey anti–rat IgG antibody 1:800 for IF (Jackson ImmunoResearch, 712-165-153), Alexa Fluor 647–conjugated donkey anti–goat IgG antibody 1:800 for IF (Jackson ImmunoResearch, 705-605-147), Cy3-conjugated donkey anti–rabbit IgG antibody 1:800 for IF (Jackson ImmunoResearch, 711-165-152), Cy3-conjugated donkey anti–mouse IgG antibody 1:800 for IF (Jackson ImmunoResearch, 715-165-151), Cy3-conjugated donkey anti–guinea pig IgG antibody 1:800 for IF (Jackson ImmunoResearch, 706-165-148), Cy3-conjugated donkey anti–rat IgG antibody 1:800 for IF (Jackson ImmunoResearch, 712-165-153), Alexa Fluor 488–conjugated donkey anti–mouse IgG antibody 1:800 for IF (Jackson ImmunoResearch, 715-545-150), Alexa Fluor 488–conjugated donkey anti–guinea pig IgG antibody 1:800 for IF (Jackson ImmunoResearch, 706-545-148), Alexa Fluor 488–conjugated donkey anti–rabbit IgG antibody 1:800 for IF (Jackson ImmunoResearch, 711-545-152), donkey anti–mouse IgG antibody (Jackson ImmunoResearch, 715-005-151), donkey anti–rat IgG antibody (Jackson ImmunoResearch, 712-005-153), donkey anti–rabbit IgG antibody (Jackson ImmunoResearch, 711-005-152), and donkey anti–chicken IgG antibody (Jackson ImmunoResearch, 703-005-155) CF583R-conjugated secondary antibodies were made by conjugating the unlabeled secondary antibodies with CF583R succinimidyl ester (Biotium, 96084) to achieve a labeling efficiency of ~2 dyes per antibody, as previously described ( ).

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