All chemicals used were of analytical or cell culture grade. All oligonucleotides were from Metabion (Steinkirchen, Germany).
Oligonucleotides
Manufactured by Metabion
Sourced in Germany
Oligonucleotides are synthetic DNA or RNA molecules that are used in a variety of applications, including genetic research, diagnostics, and therapeutics. They are typically short, single-stranded nucleic acid sequences that can be designed to target specific genes or regions of the genome.
Automatically generated - may contain errors
Lab products found in correlation
T4 polynucleotide kinase, by Thermo Fisher Scientific (2 mentions)
Infinite 200, by Tecan (1 mentions)
Lipofectamin 2000, by Thermo Fisher Scientific (1 mentions)
Qiaquick kit, by Qiagen (1 mentions)
Plasmid preparation kits, by Qiagen (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Q5 polymerase, by New England Biolabs (1 mentions)
Dpni, by New England Biolabs (1 mentions)
Nebuilder hifi dna assembly cloning kit, by New England Biolabs (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (1 mentions)
Opti fluor, by PerkinElmer (1 mentions)
Hotstartaq master mix kit, by Qiagen (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
γ 33p atp, by PerkinElmer (1 mentions)
Viia 7 system, by Thermo Fisher Scientific (1 mentions)
Gapdh hs99999905 m1, by Thermo Fisher Scientific (1 mentions)
γ 32p atp, by Hartmann Analytic (1 mentions)
γ 33p atp, by Hartmann Analytic (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
27 protocols using oligonucleotides
1
Analytical Reagents and Oligonucleotides for Research
2
miRNA-Binding Site Luciferase Assay
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Oligonucleotides of 50 to 60 base pair length (Metabion, Martinsried, Germany) containing specific miRNA‐binding sites (sequences shown in Supplementary Table S3 ) were cloned into the 3′ untranslated region (UTR) of luciferase in a reporter plasmid (pMIR‐REPORTTM miRNA Expression Reporter Vector System Ambion/Applied Biosystems/ThermoFisher Scientific) as previously described 37 . Pre‐miRsTM (Ambion/Applied Biosystems/ThermoFisher Scientific) were transfected into HeLa cells with Lipofectamin2000 (Invitrogen, Karlsruhe, Germany) along with the luciferase plasmid containing the predicted binding site of the respective miRNA. A control plasmid coding for beta‐galactosidase without any known miRNA‐binding site in its 3′UTR was used for normalization of the luciferase signal; 200 ng of each plasmid and 25 nmol of pre‐miRNA were applied to transfect 8x104 HeLa cells. Luciferase and beta‐galactosidase were measured 24 h after triple transfection (see above) using the Dual Light Luciferase Assay from Applied Biosystems/ThermoFisher Scientific and the Infinite 200 from Tecan.
3
Construction of pBW131 Plasmid Containing hfq
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All DNA manipulations, restriction digestions, ligations and transformations were performed using standard genetic and molecular techniques [89 ]. The plasmids used in this work are listed in S1 Table . Oligonucleotides used for PCR and sequencing were purchased from Metabion and are also listed in S1 Table . Plasmid DNA was isolated using Qiagen plasmid preparation kits. DNA-modifying enzymes and restriction enzymes were purchased from New England Biolabs. PCRs were performed in a 100 μl volume for 29 cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs). Purification of PCR products was routinely performed using the QIAquick kit (Qiagen). The constructed plasmid was sequenced by the in-house facility. To generate plasmid pBW131, the entire hfq gene along with 847 nt upstream region and 42 nt downstream region was amplified by PCR using primers V75 and V80 and cloned into the BamHI site of pHSG575.
4
Plasmid Construction via NEBuilder
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Oligonucleotides were synthesised by Metabion and Sigma. To construct the plasmids, DNA fragments were amplified by PCR and assembled by NEBuilder HiFi DNA Assembly Cloning Kit (NEB, E5520S).
To construct VHp770, DNA fragments were amplified by PCR using VHp277 as a template as well as sets of primers VTK69 and VTK133 or VTK19 and VTK132. To S1. The forward primer introduced the desired amino-acid substitution in its unbound 5′ region. After PCR with Q5 polymerase (NEB), the product was treated with DpnI (NEB) to remove the template plasmid, purified trough a PCR purification column (Omega), phosphorylated with PNK (NEB) and ligated (NEB). The mixture was transformed into E. coli MC1061 and the resulting plasmids were verified by sequencing (Eurofins).
To construct VHp770, DNA fragments were amplified by PCR using VHp277 as a template as well as sets of primers VTK69 and VTK133 or VTK19 and VTK132. To S1. The forward primer introduced the desired amino-acid substitution in its unbound 5′ region. After PCR with Q5 polymerase (NEB), the product was treated with DpnI (NEB) to remove the template plasmid, purified trough a PCR purification column (Omega), phosphorylated with PNK (NEB) and ligated (NEB). The mixture was transformed into E. coli MC1061 and the resulting plasmids were verified by sequencing (Eurofins).
5
Protocol for siRNA Delivery Complexes
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Complexes of nucleic acid and the CPP carriers were formed in MQ water, at N/P = 2 (nitrogen/phosphate ratio 2), as detailed in10 (link). Briefly, the complexes were formed in 100 µL of MQ, allowed to incubate for 40 minutes, after which 100 µL of 10% glucose was added and immediately injected (final injection in 200 µL in 5% glucose). The standard injected nucleic acid dose was 1.0 mg/kg.
The siRNA sequences are from the ref. 16 (link) as follows: siTNFsense 5′-pGUCUCAGCCUCUUCUCAUUCCUGct-3′, siTNFantisense 5′-AGCAGGAAUGAGAAGAGGCUGAGACAU-3′, where the RNA bases are uppercase, DNA bases are lowercase, and “p” is a 5′-phosphate. The oligonucleotides were ordered from Metabion, Germany.
The siRNA sequences are from the ref. 16 (link) as follows: siTNFsense 5′-pGUCUCAGCCUCUUCUCAUUCCUGct-3′, siTNFantisense 5′-AGCAGGAAUGAGAAGAGGCUGAGACAU-3′, where the RNA bases are uppercase, DNA bases are lowercase, and “p” is a 5′-phosphate. The oligonucleotides were ordered from Metabion, Germany.
6
qPCR Analysis of Adipose Tissue
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Gene expression levels of target genes in adipose tissue compartments were determined by reverse transcription of isolated RNA and subsequent real-time PCR. Differences and changes in gene expression levels were calculated applying the delta-delta Ct method. The applied murine primer sequences are summarized in Table 3 .
Expression levels of target genes were normalized to gene expression of murine GAPDH (isoform 1; NCBI Reference Sequence: NM_001289726.1) using a well-established primer pair that has been successfully applied for valid gene expression normalization in adipocytes in previous studies [13 (link)].
All oligonucleotides were purchased from Metabion, Martinsried, Germany.
Expression levels of target genes were normalized to gene expression of murine GAPDH (isoform 1; NCBI Reference Sequence: NM_001289726.1) using a well-established primer pair that has been successfully applied for valid gene expression normalization in adipocytes in previous studies [13 (link)].
All oligonucleotides were purchased from Metabion, Martinsried, Germany.
7
Methotrexate Transport Assay Protocol
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Restriction endonucleases and T4 ligase were obtained from Fermentas and New England Biolabs, Pfu polymerase was provided by Stratagene. Oligonucleotides were ordered from Metabion International AG and Biological Research Center of Szeged. [3H]methotrexate ([3H]MTX; 25.9 Ci/mmol) was obtained from Moravek Biochemicals. The anti-DMRP polyclonal antiserum pAB7655 was raised against a synthetic peptide corresponding to amino acids 209–222 of DMRP (ZYMED Laboratories Inc.) as described previously [17 (link)]. Secondary HRP-conjugated anti-rabbit antibodies were purchased from Jackson ImmunoResearch. Nitrocellulose membrane filters (HWAP00250) were obtained from Millipore and the scintillation fluid (Opti-fluor) from PerkinElmer. All other compounds were obtained from Sigma Aldrich. Methotrexate (MTX) was dissolved in DMSO, the final concentration of DMSO in the assay buffer was kept less than 0.1% in transport and less than 1% in ATPase experiments.
8
TRPA1 Promoter Sequence Amplification
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Amplification of TRPA1 promoter target sequences of the purified bisulfite-converted DNA was conducted in 2 different fragments, as described previously [42 (link)]. The first promoter fragment of TRPA1 was amplified using the forward primer 5′-GTTTGTATTAGATAGTTTTTTTGTTTG-3′ and the reverse primer 5′-TCCTACAAACCTATATTTCCCAC-3′, the second fragment via the forward primer 5′-GGGGTAGGGTAAGGGGTTTT-3′ and the reverse primer 5′-TACACACACCCCAAAACTTACAAC-3′, using touchdown PCRs [83 (link)] with starting temperatures of 65 °C. Oligonucleotides applied as primers were ordered from Metabion (Steinkirchen, Germany), PCRs were performed on a C1000 TM Thermal Cycler (BIO-RAD, Hercules, CA, USA) using the HotStarTaq® Master Mix Kit (QIAGEN, Hilden, Germany).
9
Radiolabeled Oligoduplex Substrate Preparation
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Oligoduplex substrates used in this study are listed in Table 1 . All oligonucleotides were purchased from Metabion (Germany). Radioactive labeling was performed with [γ-33P]ATP (PerkinElmer) and T4 polynucleotide kinase (Thermo Fisher Scientific). Oligoduplexes were assembled by annealing the corresponding radiolabeled and unlabeled strands. Construction of expression vectors, protein expression and purification are described in Supplementary Materials and Methods .
10
Quantitative RT-PCR Analysis of Gene Expression
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RT-qPCR was performed as described previously [22] (link), and on the ViiA™ 7 System (Life Technologies GmbH, Darmstadt, Germany), using TaqMan probes: HO-1 (Hs01110250_m1), GAPDH (Hs99999905_m1) and HCV (Pa03453408_s1). Oligonucleotides were obtained from Metabion International AG (Martinsried, Germany) and are summarized in Table 1 .
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