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Novagen pet28a vector

Manufactured by Merck Group
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Sourced in Germany

The Novagen pET28a(+) vector is a bacterial expression vector designed for the high-level expression of recombinant proteins in E. coli. It features a T7 promoter system, a kanamycin resistance marker, and a multiple cloning site for the insertion of target genes.

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Lab products found in correlation

Primestar max dna polymerase, by Takara Bio (1 mentions) Pet28a, by Merck Group (1 mentions) Bradford assay, by Bio-Rad (1 mentions) A gblock gene fragment, by Integrated DNA Technologies (1 mentions) Gblock gene fragment, by Integrated DNA Technologies (1 mentions)

6 protocols using novagen pet28a vector

1

Cloning and Expressing Glycoside Hydrolases

Cited 2 times
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Genes encoding ClAgl29A (EKB48091.1) and ClAgl29B
(EKB48090.1), whose locus_tags were B879_03288 and B879_03287, respectively,
were synthesized at Eurofins Genomics K.K. (Tokyo, Japan). The genes
were amplified by PCR and cloned into the Novagen pET28a vector (Merck;
Darmstadt, Germany) using an iVEC3 in vivo E. coli cloning system.30 (link) The gene encoding TmaFuc
(TM0306; nucleotide 326116 to 327465 of AE000512.1) in T. maritima NBRC 100826T genomic DNA was
amplified by PCR and cloned into pET28a using NdeI and HindIII restriction sites. Genes encoding
Caka_0074, Caka_0506, Caka_0522, Caka_0523, and Caka_0536 were cloned
based on the C. akajimensis DSM 45221
complete genome (CP001998.1): 79209..80672, 649949..651400, 670716..672635,
672662..674473, and 688656..690170, respectively. The regions were
amplified by PCR using genomic DNA of C. akajimensis JCM 23193T as a template which was provided by RIKEN
BRC through the National BioResource Project of MEXT, Japan. The PCR
products were cloned into pET28a using a T5 exonuclease DNA assembly
method.31 (link) PrimeSTAR MAX DNA Polymerase
(Takara Bio Inc., Shiga, Japan) was used for all PCR amplifications,
and reaction conditions were followed according to the attached protocol.
The primers used for the PCR are listed in Table S2.
Shishiuchi R., Kang H., Tagami T., Ueda Y., Lang W., Kimura A, & Okuyama M. (2022). Discovery of α-l-Glucosidase Raises the Possibility of α-l-Glucosides in Nature. ACS Omega, 7(50), 47411-47423.
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2

Cloning and Expression of XYR1 Variants

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A 2945 bp-fragment comprising the T7 promoter, the lac operator, and the coding region of xyr1 fused with a C-terminal six-histidine tag was chemically synthesized (GeneArt®, part of Life Technologies, Paisley, UK) with codon optimization for Escherichia coli. This expression cassette was cloned into the Novagen® pET28a vector (Merck, Darmstadt, Germany) using BglII/NotI restriction sites and yielding pTS1. Likewise, the mutated gene version, xyr1A824V, bearing the A824V transition, was chemically synthesized and cloned into pET28a, yielding pTS2. Insertion of the correct fragments into both expression vectors was confirmed by restriction profile analysis and automated sequencing (LGC Genomics, Berlin, Germany).
Mello-de-Sousa T.M., Gorsche R., Jovanović B., Mach R.L, & Mach-Aigner A.R. (2022). In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei. Journal of Fungi, 8(12), 1254.
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3

Expression and Purification of OsCYP21-4

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Expression and purification of recombinant OsCYP21-4 were carried out using the Novagen pET28a vector according to the supplier's protocols (EMD Millipore, Darmstadt, Germany). OsCYP21-4 was cloned into pET28a and sequenced. The OsCYP21-4 construct was transformed into Escherichia coli BL21 (DE3) for expression of His-tagged OsCYP21-4, and recombinant protein was purified on nickel-NTA agarose columns. Finally, the concentration and purity of OsCYP21-4-His protein were determined using the Bradford assay (Bio-Rad) and SDS-PAGE analysis.
Lee S.S., Park H.J., Jung W.Y., Lee A., Yoon D.H., You Y.N., Kim H.S., Kim B.G., Ahn J.C, & Cho H.S. (2015). OsCYP21-4, a novel Golgi-resident cyclophilin, increases oxidative stress tolerance in rice. Frontiers in Plant Science, 6, 797.
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4

Cloning DHFR2-αB7-H3 scFv Fusion Protein

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A gBlock Gene Fragment coding for DHFR2 – αB7-H3 scFv fusion protein was ordered from Integrated DNA Technologies (IDT) and cloned into the Novagen pET28a(+) vector (EMD Millipore, Cat: 69864–3) via the NcoI and XhoI restriction sites. The sequence for the αB7-H3 scFv is a humanized version of the 8H9 scFv that was affinity matured by yeast surface display.31 (link) Of note, the DHFR2 – αB7-H3 scFv construct has a myc tag and Flag tag to enable detection by flow cytometry for various applications.
Mews E.A., Beckmann P., Patchava M., Wang Y., Largaespada D.A, & Wagner C.R. (2022). Multivalent, Bispecific αB7-H3-αCD3 Chemically Self-Assembled Nanorings Direct Potent T cell Responses Against Medulloblastoma. ACS nano, 16(8), 12185-12201.
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5

DHFR2-Fn3 fusion protein expression

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A gBlock Gene Fragment coding for the DHFR2-Fn3 clone C5 fusion protein was ordered from Integrated DNA Technologies (IDT) and cloned into the Novagen pET28a(+) vector (EMD Millipore, Cat: 69864–3) via NcoI and XhoI restriction sites. Separately, Fn3 clones B22 and NT replaced clone C5 using designed NheI and BamHI restriction sites flanking the Fn3 sequence region. Notably, the DHFR2-Fn3 proteins contain an N-terminal MYC epitope tag and C-terminal polyhistidine tag to facilitate detection via flow cytometry and purification via immobilized metal affinity chromatography (IMAC), respectively.
Csizmar C.M., Petersburg J.R., Perry T.J., Rozumalski L., Hackel B.J, & Wagner C.R. (2018). Multivalent Ligand Binding to Cell Membrane Antigens: Defining the Interplay of Affinity, Valency, and Expression Density. Journal of the American Chemical Society, 141(1), 251-261.
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6

Cloning DHFR Fusion Proteins

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gBlock Gene Fragments coding for the DHFR2-Fn3 and mSA-DHFR2 fusion proteins were ordered from Integrated DNA Technologies (IDT) and cloned into the Novagen pET28a(+) vector (EMD Millipore, Cat: 69864-3) via NcoI and XhoI restriction sites. Notably, the DHFR2-Fn3 fusion proteins contain an N-terminal MYC epitope tag and C-terminal polyhistidine tag to facilitate detection via flow cytometry and purification via immobilized metal affinity chromatography (IMAC), respectively. Similarly, the mSA-DHFR2 fusion proteins contain a C-terminal FLAG epitope tag to enable flow cytometric detection.
Csizmar C.M., Petersburg J.R., Hendricks A., Stern L.A., Hackel B.J, & Wagner C.R. (2018). Engineering Reversible Cell-Cell Interactions with Lipid Anchored Prosthetic Receptors. Bioconjugate chemistry, 29(4), 1291-1301.
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