The hGal-3 coding sequence gene was optimized for Escherichia coli expression, synthesized and cloned directly into pUCIDTKan vector by the company Integrated DNA Technologies (IDT, Leuven, Belgium) and named pUCIDTKan-Gal-3. The coding sequence for the carbohydrate-recognition domain (CRD) was amplified from the vector pUCIDTKan-Gal-3 using the following primers: 5′-CCACCGGCCATATGGGCGCACCCGCTGGACC-3′ (NdeI site) and 5′-CAGGAAACAGCTATGAC-3′ (M13 reverse) and cloned into the vector pET41a (Novagen, Merck, Madrid, Spain), between NdeI and XhoI restriction sites, and named pET41-Gal-3C. This vector contains the coding sequence for the CRD from Gly108 up to the C-terminus of Gal-3 without any tag.
Pet 41a
Manufactured by Merck Group
Sourced in Spain, Japan, Germany, United States
The PET-41a is a laboratory equipment designed for the analysis and characterization of various materials. It is a versatile instrument that can be used for a range of applications. The core function of the PET-41a is to provide accurate and reliable data on the physical and chemical properties of the samples under investigation. This information can be used to support research, development, and quality control processes in various industries.
Automatically generated - may contain errors
Lab products found in correlation
Pfastbac1, by Thermo Fisher Scientific (1 mentions)
Revertra ace, by Toyobo (1 mentions)
Isogen, by Nippon Gene (1 mentions)
Bl21 de3 cells, by Merck Group (1 mentions)
Pet 41a, by New England Biolabs (1 mentions)
Wizard plus sv minipreps dna purification system, by Promega (1 mentions)
Novablue, by Merck Group (1 mentions)
Pshai, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Kod dna polymerase, by Toyobo (1 mentions)
7 protocols using pet 41a
1
Cloning the Gal-3 Carbohydrate-Recognition Domain
2
Cloning and Expression of MjgC1qR in Kuruma Shrimp
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Total RNA was extracted from adult Kuruma shrimp (10.1 cm of total length and 8.1 g of body weight) using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. First-strand cDNA was synthesized with 5 μg of total RNA using ReverTra Ace (Toyobo, Shiga, Japan) and was kept at − 30 °C until use. The obtained cDNA was used as a template for amplifying the MjgC1qR gene without a stop codon using MjgC1qR-FW-BamHI and MjgC1qR-RV-XhoI-NoStop. The final PCR products were cloned into pET-41a( +) (MERCK Japan) to fuse the gene with a polyhistidine-tagged sequence at the C-terminus (designated pET41-MjgC1qR-His), and positive clones were selected and sequenced. MjgC1qR-His was amplified from pET41-MjgC1qR-His using MjgC1qR-FW-BamHI and pET41-RV and cloned into pFastbac-1 (Thermo Fisher Scientific K. K, Tokyo, Japan), and the product was named pFB-MjgC1qR-His. The sequence was confirmed again via DNA sequencing. All the primers (including their sequences) are listed in Table 2 .
3
Recombinant BVDV E2 Protein Production
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The partial E2 gene (492 bp corresponding to the N-terminal 164 amino acids) of BVDV 1 Nose strain with N-terminal and C-terminal 6× histidine tags was synthesized by using codon
optimization (Eurofins Genomics, Tokyo, Japan). This partial E2 fragment was restriction digested and cloned into pET-41a expression vector (Merck, Darmstadt, Germany). The resulting
plasmid, pET-41a/E2, was used to transform E. coli BL21 (DE3) cells (Merck). To induce the expression of recombinant BVDV E2 protein (rE2),
isopropyl-β-d-thiogalactopyranoside (final concentration, 1.0 mM) was added to the culture, which was then incubated for 6 hr at 30°C. Inclusion bodies containing rE2 were sonicated and
solubilized in phosphate-buffered saline (PBS) containing protein denaturants (8 M urea and 10 mM β-mercaptoethanol). rE2 was affinity purified on Co2+-charged TALON resin (Takara
Bio) under denaturing conditions, according to the manufacturer’s instructions. This was followed by buffer exchange to remove denaturants. The resulting rE2 protein was used as the antigen
for ELISA and the production of monoclonal antibodies (mAbs).
optimization (Eurofins Genomics, Tokyo, Japan). This partial E2 fragment was restriction digested and cloned into pET-41a expression vector (Merck, Darmstadt, Germany). The resulting
plasmid, pET-41a/E2, was used to transform E. coli BL21 (DE3) cells (Merck). To induce the expression of recombinant BVDV E2 protein (rE2),
isopropyl-β-d-thiogalactopyranoside (final concentration, 1.0 mM) was added to the culture, which was then incubated for 6 hr at 30°C. Inclusion bodies containing rE2 were sonicated and
solubilized in phosphate-buffered saline (PBS) containing protein denaturants (8 M urea and 10 mM β-mercaptoethanol). rE2 was affinity purified on Co2+-charged TALON resin (Takara
Bio) under denaturing conditions, according to the manufacturer’s instructions. This was followed by buffer exchange to remove denaturants. The resulting rE2 protein was used as the antigen
for ELISA and the production of monoclonal antibodies (mAbs).
4
Recombinant Expression of Dictyocaulus viviparus Protein
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D. viviparus-PMY was recombinantly expressed in One Shot® BL21 Star™ (DE3) chemically competent E. coli (Life Technologies, Germany) as a glutathione-S-tranferase (GST)-fused protein using the expression vector pET-41a (Merck Millipore, Germany). After purification as previously described by Strube et al. [13 (link)], rPMY was stored at −80°C in sterile 50 mM Tris buffer (pH 8.0) until use.
5
Construction of pET-41 GST-cpzA3H Plasmid
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To construct the pET-41 glutathione S-transferase (GST)-cpzA3H plasmid for expression in bacteria, a DNA fragment of the cpzA3H open reading frame region was amplified by polymerase chain reaction (PCR) using the primer set GST-A3H (SacII_noEK)(+) and A3H (stop) XhoI (–) (Supplementary Table S1 ). The DNA fragment was cloned into pET-41a(+) (Merck Millipore), which has an N-terminal GST-HIS tag and a thrombin cleavage site. Thrombin cleavage of the GST-cpzA3H protein leaves a dipeptide (glycine-serine) preceding the N-terminus of cpzA3H. The mammalian expression plasmids of pTR600 without an insert (37 (link)) or containing a fragment for an amino-terminal FLAG-tagged cpzA3H wild-type (WT) (38 (link)) were used. Mutant cpzA3H expression plasmids were constructed by oligonucleotide-directed PCR as previously described (38 (link)) using the appropriate primer set listed in Supplementary Table S1 . The sequences of both the insert and the boundary regions for the cpzA3H expression plasmids were verified by conventional DNA sequencing methods.
6
Overexpression of FolE protein from Rickettsia monacensis in E. coli
7
Cloning and Mutagenesis of HIV-1 Protease
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The full-length PR region (2253–2549) was amplified by nested PCR using KOD DNA Polymerase (TOYOBO, Osaka, Japan). The primer set used for amplification was as follows: 5′-ATATACATATGCCTCAGAT CACTCTTTGG and 5′-TG GTGCTCGAGTTACTAAAAATTTAAAGTGCAGCC. Subsequently, the PCR product was inserted into pET-41a(+) (Merck Millipore, Billerica, MA, USA) using NdeI and XhoI restriction enzymes and a DNA Ligation Kit ver. 2.1 (Takara Bio Inc.). Mutagenesis was performed to obtain an inactive D25N PR mutant.
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