By using the SuperScript III One-Step RT-qPCR System with the Platinum Taq High Fidelity Kit (Invitrogen/ThermoFisher Scientific) and the primers (Supplementary Table 3 ), rat full-length Dnmt3a cDNA or Oct1 cDNA was synthesized and amplified from total RNA of rat DRG. A Dnmt3a shRNA duplex corresponding to bases 2576-2594 from the open reading frame of rat Dnmt3a mRNA (GenBank accession number NM_001003958) was designed. A mismatch shRNA with a scrambled sequence and no known homology to a rat gene (scrambled shRNA) was used as a control. shRNAs were synthesized and were amplified by using the primers listed in Supplementary Table 3 . Fragments harbouring Dnmt3a, Oct1, Dnmt3a shRNA, and scrambled shRNA were ligated into pro-viral plasmids using using AgeI and XbaI restriction sites. The resulting vectors expressed the genes under the control of the cytomegalovirus promotor. AAV5 viral particles carrying the cDNA were produced at the UNC Vector Core (Chapel Hill, NC). AAV5-GFP and AAV5-Cre were purchased from UNC Vector Core. HSV-GFP and HSV-Dnmt3a were provided by Dr. Eric J Nestler. Oct1 siRNA (Catalogue number: sc-36120) and its negative control siRNA (catalogue number: sc-37007) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).
Platinum taq high fidelity kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Platinum Taq High Fidelity Kit is a PCR reagent designed for high-fidelity DNA amplification. It combines Platinum Taq DNA Polymerase with a proofreading enzyme to provide increased accuracy and efficiency during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 one step rt pcr system, by Thermo Fisher Scientific (3 mentions)
Superscript 3 one step rt qpcr system, by Thermo Fisher Scientific (2 mentions)
Aavpro purification kit, by Takara Bio (2 mentions)
Aavpro titration kit, by Takara Bio (2 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Sequencer 3730 dna analyzer, by Thermo Fisher Scientific (2 mentions)
Paav mcs vector, by Cell Biolabs (1 mentions)
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Spin miniprep kit, by Qiagen (1 mentions)
Quick t4 dna ligase, by New England Biolabs (1 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (1 mentions)
Platinum pfx dna polymerase, by Thermo Fisher Scientific (1 mentions)
Bspei, by New England Biolabs (1 mentions)
Pentr d topo vector, by Thermo Fisher Scientific (1 mentions)
9 protocols using platinum taq high fidelity kit
1
Rat DRG RNA Expression Analysis
2
Elf1 cDNA Cloning and AAV Packaging
3
Generating Viral Vectors for Gene Expression
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Full‐length Pou4f3 or Raly cDNA were respectively amplified from mouse DRG RNA by using the SuperScript III One‐Step RT‐qPCR System with the Platinum Taq High Fidelity Kit (Invitrogen/Thermo‐Fisher Scientific) and forward primers with BspEI and reverse primers with NotI restriction sites (Table S2 , Supporting Information). After double enzyme digestion, the PCR products were ligated into the BspEI/NotI sites of the proviral plasmids (University of North Carolina, Chapel Hill) to replace enhanced GFP (EGFP) and the S‐D sequence. The resulting vectors expressed the genes under the control of the cytomegalovirus promoter. AAV5 packaging of viral particles carrying the cDNA was carried out using the AAVpro Purification Kit (Takara, Mountain View, CA). The virus titer was evaluated using the AAVpro Titration Kit (Takara). AAV5‐Cre was purchased from UNC Vector Core. HSV‐Gfp and HSV‐lncRNA were constructed/provided by the McGovern Institute for Brain Research at MIT. DS‐lncRNA siRNA (Table S2 , Supporting Information) and its negative control siRNA and Ehmt2 siRNA (s168026) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA).
4
Comprehensive RNA Analysis Protocol
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Total RNA from cells was extracted using TRIzol reagent (Invitrogen, United States) as previously described [20 (link)]. The complementary DNA was synthesized with random hexamer primer using Verso cDNA Synthesis Kit (Thermo Fisher Scientific, United States). Quantitative real-time PCR (qRT-PCR) was carried out on ABI StepOne Plus real time PCR system (Applied Biosystems, United States) using SYBR mix kit (Thermo Fisher Scientific, United States) as previously described [21 (link)]. Primers for the 80 NEPC related chimeric RNAs were listed in Additional file 8 : Table S1. Touch-down PCR (TD-PCR) was carried out using Platinum Taq High Fidelity Kit Invitrogen, United States. Primers for TMPRSS2-ERG (e1e4) and TMPRSS2-ERG (e2e4) were listed in Additional file 12 : Table S5.
5
Cloning and Sequencing of BRSV SH Gene
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The coding region of the rBRSV SH gene was amplified from BRSV Snook by reverse transcriptase-PCR (RT-PCR) using Invitrogen’s SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity kit following the manufacturer’s instructions. HindIII forward primer CTTAAGCTT ATGAACAATACATC and EcoRI reverse primer CGAGAATTC GGTGCTTGATTGGT were used in the reactions; the underlined sections indicate the restriction sites. The PCR product was digested with HindIII and EcoRI and the resulting product ligated into the multiple cloning site pcDNA6 (Invitrogen) in frame with V5 and histidine tags using NEB’s Quick T4 DNA ligase and 2× Quick ligation buffer following the manufacturer’s instructions. The ligation reaction was transformed into NEB 5-α competent E.coli and grown on Luria–Bertani agar plates containing 100 µg ml−1 of ampicillin. Colonies were picked and plasmid DNA was extracted using Qiagen’s spin mini-prep kit. Plasmids were sequenced commercially by the Sanger method. A single clone with the exact nucleotide sequence of the SH gene and in frame with V5 and His tags was chosen and used in subsequent experiments.
6
Zebrafish SOD1 Knockout and Neutrophil Motility
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To make sod1 mRNA, the cDNA of sod1 was amplified from zebrafish total RNA with the following primers using a SuperScript III one-step RT-PCR system with a Platinum Taq High Fidelity Kit (Invitrogen): pcs2-sod1-F: 5′-TCTTTTTGCAGGATCCTCGCCACCATGGTGAACAAGGCCGTTT-3′, pcs2-sod1-R: 5′-GTTCTAGAGGCTCGAGTCACTGAGTGATGCCGATC-3′. The purified PCR products were then inserted into the PCS2 backbone using In-Fusion cloning. To generate mRNA, the final pcs2-sod1 construct was linearized by NotI and used for in vitro transcription with a mMESSAGE mMACHINE Sp6 kit (Life Technologies). Then, 1 nl of mixture containing 25 ng/µl sod1 knocking out construct, 35 ng/µl Tol2 transposase mRNA and 300 ng/µl sod1 mRNA was injected into the cytoplasm of embryos at one-cell stage. GFP mRNA was used as the control. Neutrophil motility was measured at 3 dpf using confocal microscopy.
7
Cloning and Packaging of Rat Oct1 cDNA into AAV5
8
Influenza C Genetic Sequence Analysis
9
Influenza C Genetic Sequence Analysis
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HEF genetic sequence analysis was performed by the CDC’s Diagnostic Development Team, Influenza Division, on influenza C–positive specimens with ample residual volume and rRT-PCR cycle threshold (CT) values ≤32. The Invitrogen SuperScript III One-Step RT-PCR System with Platinum Taq High-Fidelity kits were used for PCR amplifications. Primers are available upon request. PCR products were purified by ExoSAP-IT for the PCR Product Clean-Up kit (USB Corporation). Sequencing reactions were performed using an Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit and an Applied Biosystems Sequencer 3730 DNA Analyzer. Sequences analyzed were obtained from this study or from the National Center for Biotechnology Information’s Influenza Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/; Supplementary Figure S2 ) and were aligned using the CLUSTALW program. Phylogenetic analyses were performed using Molecular Evolutionary Genetics Analysis software (version 5.1) [26 (link)]. The evolutionary history was inferred using the neighbor-joining method [27 (link)].
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