Primers with restriction enzyme sites BamHI and EcoRI were designed with Primer Premier 5.0 (Table 1 ), and the coding region of PyasOBP2 without the signal peptide was amplified with PCR. The PCR products were ligated into the pMD®19-T vector, transformed into DH5α competent cells (TaKaRa Co., Dalian, China) and then sequenced. The correct pMD®19-T plasmids were digested by restriction enzymes (BamHI and EcoRI) (TaKaRa) for 1–2 h at 37°C, cloned into the digested pET32a vector, and then transformed into DH5α cells. The correct recombinant plasmids were transformed into BL21 (DE3) competent cells (TaKaRa). Single colonies were cultured in liquid LB (supplemented with 100 mg/ml ampicillin) overnight at 37°C.
Bl21 de3 competent cells
Manufactured by Takara Bio
Sourced in China, Japan
BL21 (DE3) competent cells are a strain of Escherichia coli (E. coli) bacteria commonly used in molecular biology and biochemistry laboratories. These cells are designed for the efficient expression of recombinant proteins. They contain the DE3 lysogen, which allows for the controlled induction of protein expression using the T7 RNA polymerase system.
Automatically generated - may contain errors
Lab products found in correlation
Dh5α competent cells, by Takara Bio (1 mentions)
Dh5α cells, by Takara Bio (1 mentions)
Pet32a, by Merck Group (1 mentions)
Montanide isa763 a vg, by Seppic (1 mentions)
Pmd19 t, by Takara Bio (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Freestyle293 expi 293 media, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Histrap column, by GE Healthcare (1 mentions)
Pet28a, by Thermo Fisher Scientific (1 mentions)
Ex taq dna polymerase, by Takara Bio (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Solarbio (1 mentions)
9 protocols using bl21 de3 competent cells
1
Cloning and Expression of PyasOBP2
2
Expression and Purification of H. parasuis OppA
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According to the encoding sequence of H. parasuis OppA protein (GenBank accession No. ACL32731.1), a pair of primers was designed to amplify a 288 bp fragment (Forward: 5′-AAAGGATCCTCAGTATTCGGTAACGACTTAGA-3′ , Reversed: 5′-AAACTCGAGACCTGTGGTTGATAATGGTT-3′ ). The fragment was cloned into pET32a (+) vector (Novagen, USA) to construct the recombinant plasmids. The expression plasmid was transformed into BL21 (DE3) competent cells (Takara, China), followed by the addition of 1 mM IPTG (GE Healthcare, USA) for induction.
3
E. coli Cloning and APP Culturing
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Escherichia coli (E. coli) DH5α competent cells and BL21 (DE3) competent cells (Takara, Japan) were used for cloning and expressing RTA proteins, respectively and were grown at 37 °C in LB medium containing 50 μg/mL kanamycin.
The APP serotype 1 reference strain Shope 4074 (APP 1, CVCC259) and APP serotype 5 reference strain L20 (APP 5b L20, CVCC263) were donated by the Shanghai Entry-Exit Inspection and Quarantine Bureau. They were used for the challenge experiment and cultured in brain heart infusion (BHI, USA) medium supplemented with 10 µg/mL NAD (Sigma-Aldrich, USA) at 37 °C for 6 h with shaking at 180 rpm.
The APP serotype 1 reference strain Shope 4074 (APP 1, CVCC259) and APP serotype 5 reference strain L20 (APP 5b L20, CVCC263) were donated by the Shanghai Entry-Exit Inspection and Quarantine Bureau. They were used for the challenge experiment and cultured in brain heart infusion (BHI, USA) medium supplemented with 10 µg/mL NAD (Sigma-Aldrich, USA) at 37 °C for 6 h with shaking at 180 rpm.
4
Pathogen isolation and expression
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Y. ruckeri YRWEL01, a fish pathogen isolated from dying channel catfish in Sichuan province of China, was cultured in Brain-Heart Infusion (BHI) medium at 28°C and stored at our laboratory (17 (link)). Escherichia coli strains DH5α and BL21 (DE3) competent cells (Takara; Dalian, China) served as cloning and protein expression host, respectively. Both strains were grown in Luria-Bertani medium containing 100 μg/ml of ampicillin (Amp) at 37°C. Plasmids pMD19-T (Takara) and pET32a (+) (Merck, Germany) served as cloning and expression vectors, respectively. Montanide™ ISA763 A VG (Seppic, France) was selected for use as an adjuvant for the experiment.
5
Sourcing and Storing Viral Strains
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BL21(DE3) competent cells were purchased from TaKaRa Biomedical Technology (Beijing, China). PK-15, human embryonic kidney 293T (HEK293T), Marc145, and MDCK cells were kept in our lab. PCV2 strain HN-LB-2016, PRRSV strain BJ-4, CSFV strain Shimen, PRV strain Tangyin/Henan, PEDV strain CH_hubei_2016, and PPV reference strain 7909 were stored in the Henan Provincial Key Laboratory of Animal Immunology. SIV strain A/swine/Henan/1/2010 was stored in South China Agricultural University. The sources and GenBank accession numbers of these viruses are listed in Table 3 .
6
Cell Line Maintenance Protocols
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HEK-293T cells were purchased from American type culture collection (ATCC) and maintained in D10 medium – Dulbecco’s Modified Eagle Medium (DMEM, Corning) supplemented with 10% fetal bovine serum (GeminiBio) and 1% penicillin/streptomycin/L-glutamine (GeminiBio). HeLa-ACE2/TMPRSS2 cells were a generous gift from Dr. Jesse Bloom at the Fred Hutchinson Cancer Research Center and were maintained in D10 media. Expi-293F cells were purchased from Thermo Fisher and maintained in Freestyle293/Expi-293 media (2:1, v/v, Thermo Fisher) in polycarbonate shaking flasks (Triforest Labware). Stellar™ and BL21(DE3) competent cells were purchased from Takara Bio and Thermo Fisher, respectively.
7
Cloning and Purification of SlitOBP1
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By using gene-specific primers with the restriction enzyme sites (F-5'-CGCGAATTC ATGTTGCTGTTGTTGCGCGC-3', R-5'-CCGCTCGAGTC AGCGCGCCTCAGCCTCCA-3'), the sequence encoding SlitOBP1 was amplified with ExTaq DNA polymerase (TaKaRa, Japan). By T4 DNA ligase (Takara, China), the PCR product was connected to pET28a (Invitrogen, US) and then transformed to BL21 (DE3) competent cells (Takara, China). After the identification by PCR, the positive clone was inoculated in liquid LB overnight at 37 ℃. When its OD600 reached 0.4-0.6, 1 mM Isopropyl-D-thiogalactoside (IPTG) was added to incubate for another 12 h at 28 ℃. The total protein was purified from the supernatant by affinity chromatography using HisTrap columns (GE Healthcare). After dialysis in Tris-HCl (pH=7.4) overnight, the Bovine Enterokinase was used to remove the His-tag. The purified protein was collected and examined by 12% SDS-PAGE and Western blot. Bradford method was used to determine protein concentration 32 (link).
8
Purification and Characterization of Recombinant FUT3
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The constructed prokaryotic expression recombinant vector pET-28a-FUT3 plasmid was transformed into BL21 (DE3) competent cells (Takara Biomedical Technology). PET-28a-FUT3 bacteria induced by IPTG (Beijing Solarbio Science& Technology Co., Ltd., China) were sonicated at 75% power until the solution was clear and transparent. His-tagged FUT3 protein purification was performed with a His-tag purification nickel column kit (Beijing ComWin Biotech Co., Ltd., China). His pull-down assays were performed after incubating 10 μg His-FUT3 and Ni-NTA agarose (QIAGEN, Hilden, NRW, Germany). The protein was detected by Western blot. The protein complexes were analyzed and identified by LC-MS/MS protein profiling.
9
Cloning and Expressing RTA Proteins
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Escherichia coli (E. coli) DH5α competent cells and BL21 (DE3) competent cells (Takara, Japan) were used for cloning and expressing RTA proteins, respectively and were grown at 37°C in LB medium containing 50 μg/mL kanamycin.
The APP serotype 1 reference strain Shope 4074 (APP 1, CVCC259) and APP serotype 5 reference strain L20 (APP 5b L20, CVCC263) were donated by the Shanghai Entry-Exit Inspection and Quarantine Bureau. They were used for the challenge experiment and cultured in brain heart infusion (BHI, USA) medium supplemented with 10 µg/mL NAD (Sigma-Aldrich, USA) at 37°C for 6 h with shaking at 180 rpm.
The APP serotype 1 reference strain Shope 4074 (APP 1, CVCC259) and APP serotype 5 reference strain L20 (APP 5b L20, CVCC263) were donated by the Shanghai Entry-Exit Inspection and Quarantine Bureau. They were used for the challenge experiment and cultured in brain heart infusion (BHI, USA) medium supplemented with 10 µg/mL NAD (Sigma-Aldrich, USA) at 37°C for 6 h with shaking at 180 rpm.
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