Gibson assembly

Manufactured by Integrated DNA Technologies

Gibson assembly is a molecular cloning technique used to join multiple DNA fragments in a single, isothermal reaction. It allows for the seamless assembly of DNA fragments with overlapping sequences, facilitating the construction of recombinant DNA molecules.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Facsaria 2 sorp, by BD (3 mentions) Gblock gene fragment, by Integrated DNA Technologies (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Amicon ultra centrifugal filter device, by Merck Group (1 mentions)

5 protocols using gibson assembly

Ribosome-Tethered GCaMP for Calcium Imaging

Cited 3 times

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To tether GCaMP6 and the GFP nanobody to ribosomes, GCaMP6m (Chen et al., 2013 (link)) or the GFP nanobody (Ekstrand et al., 2014 (link); Rothbauer et al., 2006 (link)) was linked to ribosomal subunit protein RPL10 through a short linker of amino acid sequence SGRTQISSSS-FEF (Heiman et al., 2008 (link)). The resultant construct is GCaMP6-RPL10 and is referred to as ribo-GCaMP for simplicity in the paper. All constructs were designed using a combination of restriction cloning, Gibson Assembly and gBlock gene fragments (Integrated DNA Technologies). For C. elegans constructs, the sequence of rpl-1 was fused to the C-terminus of GCaMP6m using an overlap PCR strategy. All regions that underwent PCR amplification were checked through sanger sequencing (GeneWitz; Elim Biopharm) following RCA-based amplification (GE Templiphi). Constructs were made into custom AAV through Stanford Vector Core:
AAV8-hSyn-GCaMP6m/f/s (Restriction Cloning: AscI & NheI)
AAV5/8-hSyn-riboGCaMP6m/f/s (Gibson Assembly + gBlock)
AAV5/8-hSyn-DIO-riboGCaMP6m/f/s (Restriction Cloning: AscI & NheI)
Linker-RL10 amino acid sequence: SGRTQISSSSFEFSSKVSRDTLYEAVREVLHGNQRKRRKFLETVELQISLKNYDPQKDKRFSGTVRLKSTPRPKFSVCVLGDQQHCD EAKAVDIPHMDIEALKKLNKNKKLVKKLAKKYDAFLASESLIKQIPRILGPGLNKAGKFPSLLTHNENMVAKVDEVKSTIKFQMKKVLC LAVAVGHVKMTDDELVYNIHLAVNFLVSLLKKNWQNVRALYIKSTMGKPQRLY

Chen Y., Jang H., Spratt P.W., Kosar S., Taylor D.E., Essner R.A., Bai L., Leib D.E., Kuo T.W., Lin Y.C., Patel M., Subkhangulova A., Kato S., Feinberg E.H., Bender K.J., Knight Z.A, & Garrison J.L. (2020). Soma-Targeted Imaging of Neural Circuits by Ribosome Tethering. Neuron, 107(3), 454-469.e6.

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Generating HCV-specific Ramos B cells

Cited 1 time

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HCV-specific Ramos B cells stably expressing gl-AR3C, AR3C, gl-HEPC74, or HEPC74 BCRs were generated as described elsewhere^{52 (link)}. In short, the 2-1261gl gene of the pRRL EuB29 2-1261gl IgG TM.BCR.GFP.WPRE plasmid^{109 (link)} was exchanged for the heavy and light chain genes of either gl-AR3C, AR3C, gl-HEPC74, or HEPC74 using Gibson assembly (Integrated DNA Technologies). Lentiviruses were produced by co-transfecting the generated expression plasmid with pVSV-g, pMDL, and pRSV-Rev into HEK293T cells using lipofectamine 2000 (Invitrogen). Two days post-transfection, HEK293T supernatant was used to transduce IgM-negative Ramos B. Seven days post-transduction, BCR-expressing B cells were FACS sorted on GFP and IgG double-positivity (i.e., BCR-expressing cells) using a FACS Aria-II SORP (BD Biosciences). B cells were expanded and cultured indefinitely. Prior to B cell binding assays and calcium flux assays, cells were selected for GFP expression to ensure similar levels if IgG (as BCR) across the cell lines (Supplemental Fig. 3b).

Capella-Pujol J., de Gast M., Radić L., Zon I., Chumbe A., Koekkoek S., Olijhoek W., Schinkel J., van Gils M.J., Sanders R.W, & Sliepen K. (2023). Signatures of VH1-69-derived hepatitis C virus neutralizing antibody precursors defined by binding to envelope glycoproteins. Nature Communications, 14, 4036.

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Engineered B Cell Receptor Expression

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The B cell specific expression plasmid was constructed by exchanging the gl2-1261 gene of the pRRL EuB29 gl2-1261 IgGTM.BCR.GFP.WPRE plasmid (McGuire et al., 2014 (link)) with heavy and light chain genes of either COVA1-18 and COVA2-15 using Gibson assembly (Integrated DNA Technologies). The production of lentivirus in HEK293T and the subsequent transduction was conducted as described elsewhere (ter Brake et al., 2006 (link)). In short, lentiviruses were produced by co-transfecting the expression plasmid with pMDL, pVSV-g and pRSV-Rev into HEK293T cells using lipofectamine 2000 (Invitrogen). Two days post transfection, IgM-negative Ramos B cells cultured in RPMI (GIBCO) supplemented with 10% fetal calf serum, streptomycin (100 μg/mL) and penicillin (100 U/mL) (RPMI++) were transduced with filtered (0.45 μm) and concentrated (100 kDa molecular weight cutoff, GE Healthcare) HEK293T supernatant. Seven days post transduction, BCR-expressing B cells were FACS sorted on IgG and GFP double-positivity using a FACS Aria-II SORP (BD biosciences). B cells were then expanded and cultured indefinitely.

Brouwer P.J., Brinkkemper M., Maisonnasse P., Dereuddre-Bosquet N., Grobben M., Claireaux M., de Gast M., Marlin R., Chesnais V., Diry S., Allen J.D., Watanabe Y., Giezen J.M., Kerster G., Turner H.L., van der Straten K., van der Linden C.A., Aldon Y., Naninck T., Bontjer I., Burger J.A., Poniman M., Mykytyn A.Z., Okba N.M., Schermer E.E., van Breemen M.J., Ravichandran R., Caniels T.G., van Schooten J., Kahlaoui N., Contreras V., Lemaître J., Chapon C., Fang R.H., Villaudy J., Sliepen K., van der Velden Y.U., Haagmans B.L., de Bree G.J., Ginoux E., Ward A.B., Crispin M., King N.P., van der Werf S., van Gils M.J., Le Grand R, & Sanders R.W. (2021). Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection. Cell, 184(5), 1188-1200.e19.

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Engineered B Cell Expression Plasmid

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The B cell specific expression plasmid was constructed by exchanging the gl2-1261 gene of the pRRL EuB29 gl2-1261 IgGTM.BCR.GFP.WPRE plasmid^{70 (link)} with the variable heavy and light chain genes of 1C12, 3C9, PGT121 and COVA2-15 using Gibson assembly (Integrated DNA Technologies). The production of lentivirus in HEK293T cells and the subsequent transduction was conducted as described elsewhere^{71 (link)}. In short, lentiviruses were produced by co-transfecting the expression plasmid with pMDL, pVSV-g and pRSV-Rev into HEK293T cells using lipofectamine 2000 (Invitrogen). Two days post transfection, IgM-negative Ramos B cells cultured in RPMI10 were transduced with filtered (0.45 μm) and concentrated (100 kDa molecular weight cutoff, GE Healthcare) HEK293T supernatant. Seven days post-transduction, BCR-expressing B cells were sorted on IgG and green fluorescent protein (GFP) double-positivity using a FACS Aria-II SORP (BD Biosciences). B cells were then expanded and cultured indefinitely.

Claireaux M., Caniels T.G., de Gast M., Han J., Guerra D., Kerster G., van Schaik B.D., Jongejan A., Schriek A.I., Grobben M., Brouwer P.J., van der Straten K., Aldon Y., Capella-Pujol J., Snitselaar J.L., Olijhoek W., Aartse A., Brinkkemper M., Bontjer I., Burger J.A., Poniman M., Bijl T.P., Torres J.L., Copps J., Martin I.C., de Taeye S.W., de Bree G.J., Ward A.B., Sliepen K., van Kampen A.H., Moerland P.D., Sanders R.W, & van Gils M.J. (2022). A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike. Nature Communications, 13, 4539.

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Characterizing B-1a Cell CDR3 Specificity

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To characterize the binding specificity of the most common CDR3 sequence observed in peritoneal and splenic B-1a cells (CMRYGNYWYFDVW, V11-D2-J1), we generated a cDNA for a single-chain variable fragment (scFv) using the original IGHV bearing this CDR3, paired with the light chain variable region from the hybridoma sequences from the study originally describing this sequence (21 ). This was constructed by gene fragment synthesis of heavy and light chain variable regions and Gibson assembly (Integrated DNA Technologies, San Diego, CA) in a construct with a flexible 15-amino acid long linker connecting the heavy and light chain domains and designated as XQ11-scFv, which is shown in Fig. 10. A His6-tag was inserted into the C-terminus of the scFv, and the fusion construct cloned into a pFUSE mammalian expression vector (Invivogen, San Diego, CA) under the control of an hEF1-HTLV promoter. This was transiently expressed in HEK293T cells cultured first in DMEM containing 4.5 g/L glucose, 10% FBS, 100 μg/ml Zeocin, and 15 μg/ml blasticidin, and then serum-free media. The culture supernatant was concentrated using an Amicon Ultra Centrifugal Filter Device (Millipore, Burlington, MA) and then used in an ELISA to evaluate binding to antigens.

Prohaska T.A., Que X., Diehl C.J., Hendrikx S., Chang M.W., Jepsen K., Glass C.K., Benner C, & Witztum J.L. (2018). Massively parallel sequencing of peritoneal and splenic B cell repertoires highlights unique properties of B-1 cell antibodies. Journal of immunology (Baltimore, Md. : 1950), 200(5), 1702-1717.

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