RT-PCR was performed to determine whether the TPH mRNA levels were directly proportional to TPH1/2 expression in the brains of the healthy,depressive model and treated rats. Total RNA was extracted from the brains using an RNeasy Plus Mini Kit (Qiagen, 74134). Reverse transcription was performed with a PrimeScript RT-PCR Kit (TaKaRa, DRR014A) using random 6 mers (random 6-base primer for the transcription of DNA into rRNA, mRNA or tRNA) as primers. The mRNA was then amplified with Recombinant Taq DNA Polymerase (TaKaRa, R001AM) using gene-specific primers for TPH1, TPH2 and 18S ribosomal RNA (18s rRNA; used as an internal reference and positive control for RT-PCR). The primer sequences and size of the target sequences for RT-PCR are listed in Table 1 . TPH1 and TPH2 primers were designed using the Primer Premier 5 software and were designed to span across exons and introns to exclude potential DNA contamination. The PCR was initiated at 95°C for 5 min, and the amplifications were performed with 38 cycles at 95°C for 30 s (denaturation), 60°C for 30 s (annealing), and 72°C for 1 min (extension), followed by an extra extension step at 72°C for 10 min. The PCR products were then visualized with 1% agarose gels stained by Gold View (Solarbio, G8142) and viewed in a fluorescent/chemiluminescent detector (Tanon, 4500).
Recombinant taq dna polymerase
Manufactured by Takara Bio
Sourced in Japan
Recombinant Taq DNA polymerase is a thermostable DNA polymerase enzyme derived from the bacteria Thermus aquaticus. It is used for DNA amplification and sequencing applications.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt pcr kit, by Takara Bio (1 mentions)
Goldview, by Solarbio (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Redextract n amp tissue pcr kit, by Merck Group (1 mentions)
Abi prism 3730 dna sequencer, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 sequence detector system, by Roche (1 mentions)
Rnaiso reagent, by Takara Bio (1 mentions)
Reverse transcript kit, by Takara Bio (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Ion xpress plus fragment library kit, by Thermo Fisher Scientific (1 mentions)
Pcr thermal cycler dice, by Takara Bio (1 mentions)
Ion torrent pgm system, by Thermo Fisher Scientific (1 mentions)
Accuprep pcr purification kit, by Bioneer (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
6 protocols using recombinant taq dna polymerase
1
Quantifying Brain TPH mRNA Levels
2
DNA Extraction and COX1 Amplification of Corbicula uncinata
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About 500 C. uncinata were collected from the culture medium, suspended in lysis buffer (10 mM Tris–HCl, pH 8.0; 1 M EDTA, pH 8.0; 0.5% sodium dodecyl sulfate; 60 μg/ml proteinase K), and incubated at 55 °C for 12–20 h. DNA was extracted using the REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, USA). The COX1 gene was amplified with two primers listed in Additional file 1 : Table S1. Polymerase chain reaction (PCR) was conducted in a 20-µl reaction mixture containing 7.4 µl ddH2O, 10 µl 2 × PCR buffer (Mg2+, dNTP plus, Takara, Dalian, China), 0.6 µl of each primer, 0.4 µl recombinant Taq DNA polymerase (250 U/µl, Takara, Dalian, China), and 1 µl DNA template. PCR conditions were as follows: initial denaturation at 98 °C for 2 min, followed by 40 cycles at 98 °C for 10 s, 50 °C for 15 s, 68 °C for 1 min/kb, and a final extension at 68 °C for 10 min. PCR amplified products were sequenced on an ABI PRISM® 3730 DNA Sequencer (Applied Biosystems, USA).
3
Quantitative Analysis of Gene Expression
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Total RNA was prepared from cells, using RNAiso reagent (Takara). RNA was reverse transcribed into cDNA using a reverse transcript kit (Takara) according to the instructions of the manufacturer. Complementary DNA was amplified by using recombinant Taq DNA polymerase (Takara) and specific oligonucleotide primers of PGC‐1β, tumor necrosis factor receptor–associated factor 6 (TRAF6), and β‐actin (Supplementary Table 2 , available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40868/abstract ). SYBR Green–based qPCR was performed using a Roche LightCycler 480 sequence detector system (25 ).
4
Fecal Bacterial DNA Extraction and 16S Sequencing
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Fecal bacterial DNA from samples taken at various times (0, 6, 12, and 18 h) was extracted using a QIAamp DNA Stool Mini kit (Qiagen, Valencia, CA, USA), in accordance with the manufacturer’s instructions, including bead-beating twice for 5 min. The V1–V2 regions of 16S rRNA genes were amplified by polymerase chain reaction (PCR) using universal primers (8F and 338R) with barcode sequences for multiplexing sample reads. PCR was performed using a PCR Thermal Cycler Dice (Takara, Shuzu, Japan) and recombinant Taq DNA polymerase (Takara). The PCR conditions were as follows: 95 °C for 5 min; 30 cycles of 30 s at 95 °C, 1 min at 61 °C, and 40 s at 72 °C; and 5 min at 72 °C. The amplified 16S rRNA gene products were purified with an AccuPrep PCR Purification Kit (BIONEER, Daejeon, Korea).
PCR product concentrations were measured on a NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE, USA) and mixed to a constant concentration such that the total concentration was 1 mg. The Ion Xpress Plus Fragment Library Kit (Thermo Scientific, Wilmington, DE, USA) was used to form the amplicon library according to the manufacturer’s instructions, and quantification of the amplicon library was performed using a Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). The amplicon library was sequenced on an Ion Torrent PGM system (Thermo Scientific, Wilmington, DE, USA).
PCR product concentrations were measured on a NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE, USA) and mixed to a constant concentration such that the total concentration was 1 mg. The Ion Xpress Plus Fragment Library Kit (Thermo Scientific, Wilmington, DE, USA) was used to form the amplicon library according to the manufacturer’s instructions, and quantification of the amplicon library was performed using a Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). The amplicon library was sequenced on an Ion Torrent PGM system (Thermo Scientific, Wilmington, DE, USA).
5
Generating TMEM79-Deficient Mice
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Male heterozygous TMEM79-deficient (TMEM79+/−) mice on a C57BL/6NCr background were kindly provided by Dr. Takeshi Matsui and Dr. Masayuki Amagai (Laboratory for Skin Homeostasis, RIKEN, Japan). Homozygous TMEM79-deficient (TMEM79−/−) mice were generated by mating male TMEM79+/− and female wild-type mice for two generations. Wild-type, TMEM79+/−, and TMEM79−/− mice were genotyped with primers Tmem79-Intron1-F, Lar3-Universal-R, and Tmem79-Exon2-R (Supplementary Table 1 ). The recombinant Taq™ DNA Polymerase (Takara, R001) kit was used in a 10 μL PCR reaction with a thermal profile starting at 94 °C for 2 min, followed by 35 cycles at 94 °C for 15 s, 55 °C for 30 s, 72 °C for 30 s, and a final one min extension at 72 °C. All mice were maintained under SPF conditions in a controlled environment (12-h light/dark cycle with free access to food and water, 25 °C, and 50–60% humidity). All procedures were approved by the Institutional Animal Care and Use Committee of the National Institute of Natural Sciences and carried out according to the National Institutes of Health and National Institute for Physiological Sciences guidelines.
6
Viral DNA Extraction and PCR Detection
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Viral DNA was isolated using the Hirt Supernatant DNA Purification kit (GenMed Scientifics Inc., Shanghai, China), and polymerase chain reaction (PCR) was performed using recombinant Taq DNA polymerase (Takara Bio, Inc., Otsu, Japan). Primers (CMV forward 5′-ACTGCTTACTGGCTTATCG-3′ and reverse, 5′-GTCTAACTCGCTCGTCTC-3′) were used to amplify a specific 113 bp fragment for detection of the CMV sequence. Primers (γ34.5-MyD116 forward, 5′-GCGGCTCAGATTGTTCAA-3′ and γ34.5-MyD116 reverse, 5′-CGGACTGTGGAAGAGATG-3′) were used to amplify a 284 bp product that was specific for the γ34.5-MyD116 chimaera. The samples were initially denatured for 5 min at 94°C; followed by 35 cycles of denaturation at 94°C for 30 sec, re-naturation at 52°C for 30 sec and extension at 72°C for 20 sec; with a final extension at 72°C for 10 min. pVec91 or pRB4871 plasmids and PBS were used as the positive and blank controls, respectively. The PCR products were separated by size in 1% agarose gels.
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