The TmSpz-like sequence was identified from the T. molitor RNAseq (unpublished) and expressed sequence tag databases. Local-tblastn analysis was performed using the amino acid sequence of T. castaneum Spz (XP_008201191.1) as a query. The deduced amino acid sequence of TmSpz was analyzed using the BLASTx and BLASTp algorithms at the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) with nr database. The full-length target open reading frame (ORF) regions were amplified using an AccuPower Pfu PreMix (Bioneer, Daejeon, South Korea) and a MyGenie 96 thermal block (Bioneer, Daejeon, South Korea) with gene-specific primers designed using Primer 3.0 software (http://bioinfo.ut.ee/primer3-0.4.0/ ) (Table 1 ). The PCR-purified products were cloned into the T-blunt vector cloning system (Solgent Company, Daejeon, Korea). The recombinant vector was transformed into E. coli DH5α cells and sequenced using M13 primers. The full-length ORF sequence was validated after sequencing.
Mygenie 96 thermal block
Manufactured by Bioneer
Sourced in United States, Cameroon
The MyGenie 96 is a thermal block designed for accurate temperature control. It features 96 wells to accommodate various sample sizes and types. The thermal block is capable of precisely regulating temperature for efficient sample processing and analysis.
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Lab products found in correlation
M mlv reverse transcriptase, by Promega (4 mentions)
Steponeplus real time pcr, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Accupower pfu premix, by Bioneer (2 mentions)
Accupower pcr premix, by Bioneer (2 mentions)
Sybr green, by Merck Group (2 mentions)
Accupower rt premix, by Bioneer (2 mentions)
Tri reagent, by Molecular Research Center (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Biosprint 96 dna plant kit, by Qiagen (1 mentions)
Accupower pfu pcr premix, by Bioneer (1 mentions)
Reverse transcriptase, by Toyobo (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Quantitech sybr green pcr kit, by Qiagen (1 mentions)
Rneasy mini rna extraction kit, by Qiagen (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Gel doc 1000 imaging system, by Bio-Rad (1 mentions)
Accupower hotstart pcr premix kit, by Bioneer (1 mentions)
17 protocols using mygenie 96 thermal block
1
Identification and Cloning of TmSpz-like
2
Genomic DNA Extraction and PCR Analysis from Plant Materials
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BioSprint 96 DNA Plant Kit (INDICAL BIOSCIENCE, Cat. SP947057, Leipzing, Germany) was used to extract genomic DNA from plant materials. Samples were ground using Tissue LyserII (QIAGEN, Cat. 85300, Hilden, Germany), and genomic DNA was extracted according to the manual provided in the BioSprint 96 DNA Plant Kit (INDICAL BIOSCIENCE, Cat. SP947057, Leipzing, Germany). The extracted DNA was evaluated for nucleic acid quantification and quality using a Nanodrop ND 1000 spectrophotometer (ThermoFisher, Cat. ND 1000, Waltham, MA, USA). PCR analysis was performed in a My-Genie 96 Thermal block (BIONEER, Cat. A-2040-3, Daejeon, Korea) using 10 ng of DNA template and AccuPower PCR PreMix (BIONEER, Cat. K-2018, Daejeon, Korea). The PCR profile was subjected to initial denaturation at 94 °C for 5 min, denaturation for 30 s at 94 °C, annealing at 55–60 °C for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 5 min. For the PCR profile, the denaturation–annealing–extension process was performed for 35 cycles. The PCR product was confirmed using a UV transilluminator (BIORAD, Cat. 170–8070, Hercules, CA, USA) after electrophoresis on a 0.8% agarose gel containing EtBr (SIGMA, Cat. E1510, Saint Louis, MO, USA).
3
miRNA Quantification by RT-qPCR
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miRNAs were retrotranscribed using the stem–loop method as previously described37 (link) with some modifications. Briefly, 100 ng of total RNA were preheated at 70 °C during 5 min in 14 μL containing 0.07 μM of stem–loop primer. For plasma samples, cel-39 synthetic miRNA was spiked in before RNA extraction, and 4 μl of total RNA were used for retrotranscription. Retrotranscription was performed using M-MLV reverse transcriptase (Promega) and incubated in MyGenie96 Thermal Block (Bioneer) for 30 min at 16 °C, 50 min at 37 °C and 15 min at 70 °C. qPCR was performed in 25 μL with 0.05–1 μL RT product, 1U Taq DNA polymerase (Pegasus), 4 mM MgCl2, 0.2 mM dNTPs, 3 × 10−5 μL Sybrgreen (Sigma), 0.1 μM forward primer, and 0.1 μM reverse primer. The reactions were incubated in StepOne Plus Real Time PCR (Applied Biosystems) at 94 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, annealing temperature for 20 s and 72 °C for 25 s. All reactions were run in duplicate. The expression levels of miRNAs were normalized to hsa-miR-103a-3p levels and control. Primer sequences for miRNA RT-qPCR are listed in Supplemental Table 1 .
4
Total RNA Extraction and cDNA Synthesis
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The total RNA was isolated from the developmental stages, tissues, and time-course samples using a Clear-S™ Total RNA extraction kit (Invirustech Co., Gwangju, South Korea) according to the manufacturer’s instructions. The total RNA (2 μg) was used as the template to synthesize cDNA using the Oligo(dT)12–18 primer on MyGenie96 Thermal Block (Bioneer, South Korea) and AccuPower® RT PreMix (Bioneer) according to the manufacturer’s instructions. The cDNA was stored at −20°C until required.
5
Genetic Diversity Analysis of Rice Varieties
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Genomic DNA of the 187 rice varieties was extracted from the fresh young leaves of 2-week-old seedlings using a BioSprint 96 DNA Plant Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. PCR was carried out using AccuPower PCR Premix (Bioneer, Daejeon, Korea) with 10 pmol forward and reverse primers and 50–100 ng template DNA in a 20-μl total reaction volume. The primer sequences of 10 previously reported SSRG markers are listed in Table 1 . In a MyGenie 96 thermal block (Bioneer), amplifications were carried out with the following conditions: initial denaturation at 95°C for 3 min followed by 35 cycles of 45 s at 94°C, 45 s at 55–60°C, and 1 min at 72°C, and a final extension at 72°C for 5 min. Banding patterns were visualized by electrophoretic separation of the PCR products on 1.0% agarose (AGPase-L InDel, SSIIa TT/GC SNPs, and SBEI InDel) or 8.0% polyacrylamide gels (GBSSI InDel and SDBE InDel). For other SSRG markers, PCR product was digested with an appropriate restriction enzyme before the electrophoresis on 1.0% agarose gel (SBEIII C/G SNP) or 8.0% polyacrylamide gel (AGPase-S C/T and A/T SNPs, and GBSSI T/G and T/C SNPs). Detailed PCR and electrophoresis procedures for each marker are described in the references provided in Table 1 . Based on the detected banding patterns of SSRG markers, allele types were assigned to each rice variety.
6
Characterization of Atg8 Gene in Aedes albopictus
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The AaAtg8 gene sequence was retrieved by performing a local TBLASTN analysis using the Tenebrio molitor Atg8 amino acid sequence (AJE26300.1) as the query and an unpublished Ae. albopictus nucleotide database, generated from an RNA sequencing analysis, as the subject. To confirm the full-length open reading frame (ORF) sequence of AaAtg8, gene specific primers (as provided in Table 1 ) were designed using the program primer3 (http://bioinfo.ut.ee/primer3-0.4.0/ ). The target gene was amplified by the AccuPower® Pfu PCR PreMix (Bioneer, Daejeon, South Korea) on a MyGenie 96 thermal block (Bioneer, Daejeon, South Korea). The PCR product was cloned into the T-blunt vector using the T-bluntTM PCR Cloning Kit (Solgent Company, South Korea), transformed into competent E. coli (DH5α) cells, and sequenced. For domain analysis and residue functions, the expected amino acid sequence of AaAtg8 was subjected to InterProScan (https://www.ebi.ac.uk/interpro/search/sequence-search ) and BLASTP analyses. In addition, we performed a pairwise alignment of the AaAtg8 gene of the Korean and Chinese strains (XP_001652571.1) of Ae. albopictus at the nucleotide and amino acid sequence levels using the Clustal X2.1 program [56 (link)].
7
Quantifying Cellular and Plasma miRNA Levels
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Total RNA from cells, allografts, WAT, or plasma was isolated using TriReagent (Molecular Research Center, Cincinnati, OH, USA). For plasma samples, cel‐miR‐39 synthetic miRNA (20–40 fmol) was spiked in before RNA isolation. miRNAs were retrotranscribed using the stem‐loop method as previously described [8 , 17 ]. For RT, 100 ng (cells, allografts or WAT) or 4 μL (plasma o culture medium) of total RNA and 0.07 μm of stem‐loop primer were preheated (70 °C, 5 min). RT was performed using M‐MLV reverse transcriptase (Promega, Madison, WI, USA) and incubated in MyGenie96 Thermal Block (Bioneer, Daedeok‐gu, Daejeon, Republic of Korea) (30 min 16 °C, 50 min 37 °C, 15 min 70 °C).
8
Antiviral Effects of Epimedium koreanum
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RAW264.7 and HEK293T cells were grown in six-well tissue culture (TC) plates (1 × 106 cells/well) and incubated at 37 °C; the cells were treated with DMEM + 10% FBS alone (negative control), DMEM with 1000 units/mL recombinant murine/human IFN-β), DMEM with 1.0 μg/mL (10 μl/mL or 1%) Epimedium koreanum Nakai, and the cells were harvested at 0, 3, 6, 12, and 24 hpt. The total RNA from the cells was isolated using the RNeasy Mini Kit (Qiagen, Seoul, Korea), and cDNA synthesis was performed using reverse transcriptase (Toyobo, Japan). The different levels of cDNA were quantified by real-time polymerase chain reaction (PCR) using a QuantiTect SYBR Green PCR kit (Qiagen, Seoul, Korea) on a Mygenie96 thermal block (Bioneer, Korea). The PCR primers are listed in Table 2 and Table 3 .
9
Measuring Interferon Response in Cell Lines
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RAW264.7 or HEK293T cells were treated with DMEM supplemented with media alone (negative control), DMEM with 1000 units/ml rmIFN-β or rhIFN-β (positive control), or DMEM with 1.0 μg/ml CP and the cells were harvested at 0, 3, 6, 12, and 24 hpt. The total RNA isolation, cDNA synthesis, and visualization of band intensity after PCR were as previously described. Additionally, different cDNA levels from HEK293T cells were measured by quantitative RT- PCR (qRT-PCR) using a QuantiTech SYBR Green PCR kit (Qiagen, Seoul, Korea) on a Mygenie96 thermal block (Bioneer, Daejeon, Korea). The PCR primers are listed in Tables 3 and 4 .
10
Quantifying Gene Expression in Cells and Tissues
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Total RNA was isolated from cultured cells and mouse tissues, using the RNeasy RNA extraction Mini Kit (Qiagen). One microgram of total RNA was used to synthesize cDNA with RT–PCR reagent Kit (Enzynomix). Quantitative PCR was performed with gene-specific primer sets (Supplementary Table 1 ) and Mygenie 96 thermal block (Bioneer) using the SYBR Green PCR Matster Mix. Real-time PCR was carried out using the CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad). Data were normalized to the level of GAPDH expression in each individual sample.
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