A set of 11 primer pairs were synthesized using the software Primer3 (Untergasser et al., 2012 (link)) and validated in 21 accessions of Piper representing 12 different species (Piper nigrum, Piper longum, Piper arboreum, Piper argyrophyllum, Piper attenuatum, Piper betel, Piper chaba, Piper hymenophyllum, Piper trichostachyon, Piper wallichi, Piper columbrinum, and Piper sarmentosum). Genomic DNA was isolated from the leaf using the cTAB DNA extraction method. The PCR reaction consisted of 1× PCR buffer, 2.5 mM of MgCl2, 1 µM of primer, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 15 ng of template DNA in a total volume of 20 µl and cycled at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min followed by a final extension at 72°C for 10 min. The amplification products were resolved on a QIAxcel multicapillary system using QIAxcel High Resolution Kit 1200 (QIAGEN, No. 929002, New Delhi,Qiagen India Pvt. Ltd.) 50-800 bp v2.0 QX DNA size marker (QIAGEN, No. 929561, New Delhi,Qiagen India Pvt. Ltd.) and 15 bp/1,000 bp Qx alignment marker (QIAGEN No. 929521, New Delhi,Qiagen India Pvt. Ltd.) with the high-resolution run method OM700. The allelic sizes of each sample were calculated in the form of gel profiles and peaks using the QIAxcel ScreenGel software (QIAGEN, v1.5).
Qx alignment marker
Manufactured by Qiagen
Sourced in Germany
The QX Alignment Marker is a laboratory equipment product designed to aid in the alignment and calibration of the QX200 Droplet Digital PCR system. It is used to ensure accurate and consistent measurements during the digital PCR analysis process.
Automatically generated - may contain errors
Lab products found in correlation
Qiaxcel connect system, by Qiagen (2 mentions)
Dna high resolution kit, by Qiagen (2 mentions)
Qiaxcel screengel software, by Qiagen (1 mentions)
Qx dna size marker, by Qiagen (1 mentions)
Dna dilution buffer, by Qiagen (1 mentions)
Quantifast sybr green pcr kit, by Qiagen (1 mentions)
Quantitect kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lightcycler 2, by Roche (1 mentions)
Qiaxcel advanced system, by Qiagen (1 mentions)
5 protocols using qx alignment marker
1
Validation of Piper Species Primers
2
Soybean Genotyping with SSR Markers
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The DNA was extracted from 4 day old seedlings of soybean accessions in two replicates [73 (link)]. The genotyping of the soybean collection was conducted using twenty-five SSR markers (Table S1 ). These SSRs were selected based on their associations with PH (Table S1 ). PCR conditions were optimized in order to provide high efficiency and accuracy of amplification [15 (link)]. The PCR was performed in a total volume of 20 µL, comprising 20 ng of genomic DNA, 1 U of Taq polymerase, 0.2 mM of each deoxyribonucleotide triphosphate (dNTP), 10 pM of each primer, 1.5 mM of magnesium chloride (MgCl2), and a standardized 1× Taq buffer solution. Table S1 summarizes information about the chromosome positions, primers, and motifs of each SSR marker in the analysis.
The PCR products were separated on a QIAxcel Connect System for capillary electrophoresis (QIAGEN, Stockach, Germany) using a QIAxcel DNA High Resolution Kit and QX Alignment Marker (15 bp/3 kb) (Figure S3 ). The OH500 method was used to run the samples with an injection time of 20 s.
The PCR products were separated on a QIAxcel Connect System for capillary electrophoresis (QIAGEN, Stockach, Germany) using a QIAxcel DNA High Resolution Kit and QX Alignment Marker (15 bp/3 kb) (
3
Soybean Genotyping with SSR Markers
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The DNA was extracted from 4 day old seedlings of soybean accessions in two replicates [73 (link)]. The genotyping of the soybean collection was conducted using twenty-five SSR markers (Table S1 ). These SSRs were selected based on their associations with PH (Table S1 ). PCR conditions were optimized in order to provide high efficiency and accuracy of amplification [15 (link)]. The PCR was performed in a total volume of 20 µL, comprising 20 ng of genomic DNA, 1 U of Taq polymerase, 0.2 mM of each deoxyribonucleotide triphosphate (dNTP), 10 pM of each primer, 1.5 mM of magnesium chloride (MgCl2), and a standardized 1× Taq buffer solution. Table S1 summarizes information about the chromosome positions, primers, and motifs of each SSR marker in the analysis.
The PCR products were separated on a QIAxcel Connect System for capillary electrophoresis (QIAGEN, Stockach, Germany) using a QIAxcel DNA High Resolution Kit and QX Alignment Marker (15 bp/3 kb) (Figure S3 ). The OH500 method was used to run the samples with an injection time of 20 s.
The PCR products were separated on a QIAxcel Connect System for capillary electrophoresis (QIAGEN, Stockach, Germany) using a QIAxcel DNA High Resolution Kit and QX Alignment Marker (15 bp/3 kb) (
4
Quantifying Genetic Composition via PCR
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A series of templates composed of different ratios of BN and LEW DNA, including 0%, 0.1%, 0.2%, 0.4%, 0.8%, 1%, 2%, 4%, 8%, 10%, 20%, 40%, 60%, 80%, and 100% BN DNA, were prepared as templates for D10Rat25-PCR. Following PCR amplification, capillary electrophoresis was performed to resolve the products, whose sizes were determined by referencing the signals of a QX Alignment Marker and QX DNA Size Marker (QIAGEN, Valencia, CA). For each template, signal intensities stemming from BN and LEW D10Rat25 were obtained, and the ratio of BN/(BN+LEW) was derived. The real percentage of DNA was plotted against the corresponding signal ratio, and a standard curve was derived by linear regression with a correlation coefficient (R2).
5
BDNF-induced mRNA Expression Analysis
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Total RNA of MLO-Y4 with and without BDNF treatment for 24 h (n = 5) was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Then, the RNA was reverse transcribed with the Quantitect Kit (Qiagen), where first the contaminating DNA was removed by incubation with 2 µL DNA Wipeout buffer for 2 min at 42 °C. Afterwards, cDNA was diluted 1:4 with RNase-free water. For PCR, 4 µL diluted cDNA, 0.2 µL forward primer, 0.2 µL reverse primer (Table 1 ), and 0.6 µL RNase-free water were added to 5 µL Mastermix of the QuantiFast SYBR Green PCR Kit (Qiagen). The PCR was carried out in the LightCycler 2.0 (Roche) with the following conditions: 5 min denaturation at 95 °C, 40 cycles with 10 s denaturation at 95 °C and 30 s polymerization and elongation at 60 °C followed by melting curve analysis where the temperature increased stepwise from 60 to 95 °C. The melting curve proved the purity of PCR products. PCR products were additionally screened by gel electrophoresis using the QIAxcel Advanced System (Qiagen). Therefore, the samples were diluted 1:3 with DNA dilution buffer (Qiagen), attached to the QIAxcel Advanced System and compared with QX alignment marker (Qiagen). For semi-quantitative analyses, relative expression was calculated by the ΔΔ cycler threshold (Ct) method. βActin was used as reference gene.
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