Point mutations in COX2 (S565A) was generated using the In-Fusion Cloning Kits (Clontech) and human COX2 gene ORF cDNA clone expression plasmid (Catalog number: HG12036-NH, Sino Biological Inc), according to the manufacturer’s instructions, and the following primer and its reverse-complement for point mutation were used: Forward 5′-CTTTACTGCTTTCAGTGTTCCAGATCCAGAGC-3′; Reverse 5′- CTGAAAGCAGTAAAGGGACAGCCCTTCACG-3′. The COX2 WT plasmids and COX S565A plasmids were transformed in the One shot TOP 10 competent cell (Invitrogen). The bacteria were grown in Luria-Bertani (LB) plate supplemented with 50 μg/ml kanamycin at 37 °C. The bacterial cell pellets were resuspended in lysis buffer containing 50 mM Tris (PH 8.0), 500 mM NaCl (Sigma-Aldrich), complete EDTA-free protease inhibitor (Roche), DNase I (Roche) and 0.25 mM DTT before being lysed by sonication and clarified by centrifugation at 75,600 × g. Proteins were purified from the soluble fraction using nickel-NTA agarose (QIAGEN) and acetylation assay was performed.
One shot top 10 competent cell
Manufactured by Thermo Fisher Scientific
Sourced in Germany
One Shot Top 10 Competent Cells are a type of laboratory equipment used for bacterial transformation. They facilitate the efficient uptake of DNA by E. coli cells, enabling genetic manipulation and recombinant protein production.
Automatically generated - may contain errors
Lab products found in correlation
In fusion cloning kit, by Takara Bio (2 mentions)
Dnase 1, by Roche (2 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
Nickel nta agarose, by Qiagen (1 mentions)
Lysozyme, by Merck Group (1 mentions)
His spintrap column, by GE Healthcare (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Luria bertani lb, by Thermo Fisher Scientific (1 mentions)
Imidazole, by GE Healthcare (1 mentions)
Sodium chloride (nacl), by GE Healthcare (1 mentions)
Bigdye terminator cycle sequencing ready reaction kit, by Thermo Fisher Scientific (1 mentions)
Ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Peqgold gel extraction kit, by Avantor (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Plasmid midiprep kit, by Qiagen (1 mentions)
5 protocols using one shot top 10 competent cell
1
Generation and Purification of COX2 S565A Mutant
2
Mutational Analysis of SphK1 and COX2
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Single point mutations in SphK1 (R56A, R57A, R24A, R185A, D178A, and F192A) and COX2 (S565A, N181A, T564A, and S567A) were generated using the In-Fusion Cloning Kits (Clontech) and ORF cDNA expression plasmids of human SphK1 and COX2 gene (Sino Biological Inc, HG15679-NH and HG12036-NH), according to the manufacturer’s instructions. The forward and reverse primers described in Supplementary Table 4 . The SphK1 WT, SphK1 mutants, COX2 WT, and COX2 mutant plasmids were transformed in the One shot TOP 10 competent cell (Invitrogen). The bacteria were grown in Luria-Bertani (LB, Invitrogen) plates supplemented with 50 μg ml−1 kanamycin (Sigma-Aldrich) at 37 °C. The bacterial cell pellets were resuspended in lysis buffer containing 20 mM sodium phosphate (GE Healthcare), 500 mM NaCl (GE Healthcare), 20 mM imidazole (pH 7.4) (GE Healthcare), 0.2 mg ml−1 lysozyme (Sigma-Aldrich), 20 μg ml−1 DNase I (Roche), 1 mM MgCl2 (Sigma-Aldrich), and 1 mM PMSF (Sigma-Aldrich) before being lysed by sonication and clarified by centrifugation at 15,493×g for 10 min. Proteins were purified from the soluble fraction using His-Spin Trap columns (GE Healthcare) and the binding and acetylation assays were performed.
3
Cloning and Sequencing of RT-PCR Amplicons
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The RT‐PCR amplification products were visualized on an agarose gel (1·5%) and fragments of 900 bp were extracted using a peqGOLD Gel Extraction Kit (Peqlab). Afterwards, the products were cloned using the TA Cloning Kit (Life Technologies, Darmstadt, Germany) and One Shot Top 10 Competent Cells (Life Technologies). After 12 hr of incubation on ampicillin‐containing agar, colonies were picked and expanded in overnight culture at 37° for plasmid isolation with the peqGOLD Mini Prep Kit (Peqlab). Sequencing reactions were performed with the BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems v1·1) according to the manufacturer's instructions in 96‐well PCR‐plates (Kisker, Steinfurt, Germany) in a C1000 Thermal Cycler (BioRad, Munich, Germany). To avoid sequencing errors each PCR fragment was sequenced in both directions.
4
Creation of espGΔARF and ΔPAK Plasmids
5
Calreticulin Expression in Dendritic Cells
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Spleens were harvested from either B6-WT or Ik−/− animals and incubated with collagenase D (1 mg/mL, Roche Diagnostics, Indianapolis, IN) in a 37°C/5% CO2 incubator for 45 min. Cells were mashed and purified with CD11c microbeads (Miltenyi Biotec) and auto MACS (Miltenyi Biotec). Calreticulin expression was detected by western blotting.
For overexpression transfection into DCs, we used the plasmid pMSV-mCRT (a gift from Dr. Malini Raghavan, Department of Microbiology and Immunology, the University of Michigan Medical School), which contains full-length mouse wild-type calreticulin cDNA. The pMSV-mCRT plasmid was transformed into Oneshot Top 10 competent cells (Life Technologies) and transfectants were selected by ampicillin. Positive strains were expanded and purified using an Endofree Plasmid Purification kit (Qiagen). Purified pMSV-mCRT plasmids and empty vector control were transfected into DCs from WT-B6 or Ik−/− animals in a 6-well plate (at 80% confluency) using X-treme GENE HP DNA Transfection Reagent (Roche) according to the manufacturer's instruction. After incubation at 37°C for 48 h, the transfected cells were collected for assays.
For overexpression transfection into DCs, we used the plasmid pMSV-mCRT (a gift from Dr. Malini Raghavan, Department of Microbiology and Immunology, the University of Michigan Medical School), which contains full-length mouse wild-type calreticulin cDNA. The pMSV-mCRT plasmid was transformed into Oneshot Top 10 competent cells (Life Technologies) and transfectants were selected by ampicillin. Positive strains were expanded and purified using an Endofree Plasmid Purification kit (Qiagen). Purified pMSV-mCRT plasmids and empty vector control were transfected into DCs from WT-B6 or Ik−/− animals in a 6-well plate (at 80% confluency) using X-treme GENE HP DNA Transfection Reagent (Roche) according to the manufacturer's instruction. After incubation at 37°C for 48 h, the transfected cells were collected for assays.
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