The Tm0364 (KEGG ID) gene was amplified from a T. maritima cDNA library by PCR reaction using pfu DNA polymerase. The PCR product was purified using agarose gel electrophoresis and Roche PCR purification kit (Roche Diagnostics GmbH, Mannheim, Germany). Vector pET22a and the PCR product were digested using the restriction enzymes NdeI and XhoI. The resulting fragments were purified through agarose gel electrophoresis and the Roche PCR purification kit (Roche Diagnostics GmbH, Mannheim, Germany) and finally ligated using T4 DNA ligase (ThermoFisher, Waltham, MA, USA). The ligation product was used to transform the genes into electrocompetent Escherichia coli MC1061 cells. The TmAMyA gene was cloned in the same way, and it has been previously reported [43 (link)].
Pcr purification kit
Manufactured by Roche
Sourced in Germany
The PCR purification kit is a laboratory instrument designed for the purification of PCR amplicons. It removes unwanted components from the PCR reaction, such as primers, nucleotides, and enzymes, to obtain high-purity DNA fragments suitable for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Restriction enzymes, by Jena Biosciences (2 mentions)
Trypsin enzyme, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Iproof high fidelity dna polymerase, by Bio-Rad (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Nucleotrap kit, by Macherey-Nagel (1 mentions)
Abi prism 3130xl sequencer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator ver 3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Megascript sp6 polymerase kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
E coli dh5α, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Megascript rnai kit, by Thermo Fisher Scientific (1 mentions)
Pjet1.2 vector, by Thermo Fisher Scientific (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Gel extraction kit, by New England Biolabs (1 mentions)
19 protocols using pcr purification kit
1
Cloning and Expressing TmAMyA Gene
2
Molecular Identification of Dunaliella Species
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Isolation of total DNA content from the studied strains was carried out by using the DNeasy plant minikit (QIAGENE, Germany). Species-specific oligonucleotides, namely, MA1 and MA3 (without any restriction site), corresponding to the conserved regions of 5′ and 3′ termini were used to amplify 18S rDNA gene. PCR reactions were performed according to the method described by Olmos and coworkers [26 (link)]. The molecular weights of PCR-amplified products were calculated and confirmed using a gel documentation system. PCR amplicons were purified using the PCR purification kit (Roche) according to the manufacturer's instructions. Then, the purified products were sequenced by Macrogen Company (Korea). Using BLAST software, the obtained sequences were compared with those deposited in NCBI GenBank as 18S rDNA and ITS regions of different Dunaliella species.
A neighbor-joining tree was constructed using the software MEGA version 4. Evolutionary distances were computed using the maximum composite likelihood model. For analysis, 1000 bootstrap replicates were performed to assess the statistical support for the tree. Phylogenetic studies included Chlamydomonas pumilio, as the outgroup.
A neighbor-joining tree was constructed using the software MEGA version 4. Evolutionary distances were computed using the maximum composite likelihood model. For analysis, 1000 bootstrap replicates were performed to assess the statistical support for the tree. Phylogenetic studies included Chlamydomonas pumilio, as the outgroup.
3
Chromatin Immunoprecipitation Assay for NF-κB
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A ChIP assay kit was used according to the Upstate Biotechnology ChIP protocol. Briefly, 5 × 106 cells were treated with 5-Aza (20 μM) and/or TNFα (20 ng/mL) for the indicated time periods. Protein-DNA complexes were immunoprecipitated overnight at 4°C with antibodies against NF-κB p65. Antibody complexes were pulled down for 4 h with 60 μL Protein A agarose/salmon sperm DNA. Unbound chromatin in the no-antibody sample was used as input. DNA from both unbound and eluted chromatin was purified using the PCR Purification Kit (Roche, Bromma, Sweden). The immunoprecipitated DNA was quantified by real-time quantitative PCR. Amplification was performed with the default PCR setting using the following primers: 5′-ATT CCA GCC CCT GTC TGG GT-3′ (forward) and 5′-GTG CGT GTG CGC TCT CTC T-3′ (reverse). Input DNA was used as the endogenous control.
4
Plasmid DNA Extraction and Manipulation
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Plasmid DNA was extracted from E. coli with the GenEluteTMHP Plasmid miniprep purification kit, as recommended by the manufacturer (Sigma). Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturer (New England Biolabs and Promega, respectively). Oligonucleotide primers were synthesized by Eurogentec (Seraing, Belgium). PCR was performed in a T100 thermal cycler (Biorad) with the iProof high-fidelity DNA polymerase (Biorad). Amplified DNA fragments were purified with a PCR purification kit (Roche) and separated by electrophoresis in 1% agarose gels after digestion. Hydrolyzed DNA fragments were extracted from agarose gels with the NucleoTrap kit from Macherey-Nagel. All constructs were confirmed by DNA sequencing (MWG).
5
Mitochondrial COI Sequencing of Pigeons
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Mitochondrial COI sequence was analyzed for one sample of the 10 Egyptian breeds and of Japanese feral pigeons. Moreover, one sample was sequenced from the four wild pigeon species: Emerald dove (Chalcophaps indica), Oriental turtle dove (Streptopelia orientalis), Whistling green pigeon (Treron formosae), and white-bellied green pigeon (Treron sieboldii) for comparison. The primers and polymerase chain reaction (PCR) condition are the same as described by Ramadan et al. [10 ].
The amplified products were purified using PCR Purification Kit (Roche, Mannheim, Germany), and the resultant products were sequenced using the same primers and the Big Dye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the standard protocol and electrophoresed on an ABI PRISM 3130xl sequencer (Applied Biosystems). The MEGA 6 Software (https://www.megasoftware.net ) [16 (link)] was used for sequences alignment and to infer the phylogenetic relationships based on neighbor-joining [17 (link)] methods [18 (link)].
The amplified products were purified using PCR Purification Kit (Roche, Mannheim, Germany), and the resultant products were sequenced using the same primers and the Big Dye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the standard protocol and electrophoresed on an ABI PRISM 3130xl sequencer (Applied Biosystems). The MEGA 6 Software (
6
Linearized Replicon RNA Synthesis
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Replicon DNA was linearized by digestion with NotI, purified using a PCR purification kit (Roche) and 1 µg of the linearized DNA was in vitro transcribed using MEGAscript SP6 polymerase kit (Ambion) in the presence of Cap analog (Ambion). Control Luciferase SP6 DNA was linearized by digestion with XmnI, purified by gel extraction and in vitro transcribed as before. For all subsequent experiments, 2 µl of in vitro transcription was transfected.
7
Chromatin Immunoprecipitation of ERRα in MEFs
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MEFs or undifferentiated 3T3-L1 preadipocytes were cross-linked with 1% formaldehyde, and nuclei were enriched by sequential centrifugations. After sonication, immunoprecipitation was performed overnight at 4°C with an anti-ERRα rabbit antibody (Epitomics 2131) or a control anti-rabbit IgG antibody (Sigma-Aldrich) using Dynabeads (Life Technologies). Enriched DNA was then purified using the Qiagen PCR purification kit and analyzed by qPCR using a Roche LightCycler 480. Nontargeted rabbit IgG ChIP was used as a control of antibody-nonspecific binding, and two negative regions (Ctrl 1 and Ctrl 2) were used for normalization. Fold enrichment was calculated following normalization with both negative regions and IgG background (set at 1). ChIP primers are listed in Supplemental Table S1 .
8
Bacterial Cloning Protocol Using E. coli DH5α
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Materials: All material in this study were prepared from the Merck Company (Tehran, Iran). PCR purification kit, restriction enzymes, and Master Mix for PCR amplification were supplied by the Roche and Amplicon/iNtRON Company. DNA Ladder Mix, high pure agarose, and primers were prepared by Fermentase, Invitrogen, and GenFanAvaran, respectively. pJET1.2 plasmids were prepared by the Fermentase Company. In this study, E. coli DH5α was used as a bacterial strain for cloning purposes and was supplied by the Invitrogen Company.
9
RNA Extraction and dsRNA Synthesis
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RNA was extracted from Aag2 cells using TRIzol and reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen) following the manufacturer's instructions. PCR products were generated with T7 promoter sequences at either end of the fragment using the primers listed in table 1 and designated as for use in vitro. The PCR product was blunt end cloned into a pJet1.2 vector (Thermo Scientific) following the manufacturer's instructions. Cloned PCR fragments were verified by sequencing. PCR was performed on the cloned fragments and the products purified using the PCR purification kit (Roche). peGFP-C1 (Clontech) was used as a template for the amplification of control dsRNA, targeting eGFP. dsRNA was synthesized using the Megascript RNAi kit (Ambion) following manufacturer's instructions.
10
Site-Directed Mutagenesis Protocol for CIDEA Constructs
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CIDEA WT and point-mutation (R47E and R171E) constructs were previously generated as detailed in Barneda et al.27 (link). Insertion fragments were amplified by PCR (list of primers can be found in Supplementary Data 1 ), purified by PCR purification kit (Roche, 11732676001), followed by restriction enzyme digestion. Digested fragments with sticky ends were purified by gel electrophoresis and extracted by gel extraction kit (NEB, T1020S). Purified inserts were ligated to pre-digested pPyCAGIP plasmids by DNA ligase (NEB, M020S) at 16 °C overnight. Ligation product was transformed into DHFalpha competent bacterial cells and amplified. Bacteria culture was lysed, and plasmids purified by midiprep plasmid extraction kits (Invitrogen, K210004), followed by Sanger sequencing for verification.
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