For WmCSV identification, total nucleic acids were extracted from 26 watermelon samples tested by DAS-ELISA, five melons, and four wild watermelons, as described by Dellaporta et al. (1983) (link). PCR was performed using primers specific for the coat protein (CP) region of the WmCSV genome (WmA150F 5′-gtc agt atg tgg gat cca ttg c-3′ and WmA1350R 5′-gca aat acg att caa cca caa cc-3′), using 2X KAPA Taq Ready Mix (KAPA Biosystems, Boston, Massachusetts, USA). PCR was performed using a thermal cycler (Eppendorf, AG, Hamburg, Germany), according to the method described by Ali-Shtayeh et al. (2014) (link). DNA was visualized, as described by Sambrook and Russell (2001) , and photographed using a DNA documentation gel analysis system (IN GENIUS, Syngene Bio Imaging, Cambridge, UK). A DNA Ladder (100 bp RTU; Genedirex, Inc. Taoyuan, Taiwan) was used to determine the size of the amplified PCR products.
Ingenius
Manufactured by Syngene
Sourced in United Kingdom, United States
InGenius is a compact, automated liquid handling system designed for a variety of laboratory applications. It offers precise and accurate liquid handling capabilities, enabling efficient and reliable sample processing and assay setup. The core function of InGenius is to automate liquid handling tasks, providing consistency and reproducibility in various laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Kapa taq readymix, by Roche (1 mentions)
Thermal cycler, by Eppendorf (1 mentions)
Dna ladder, by GeneDireX (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Amplitaq gold polymerase, by Thermo Fisher Scientific (1 mentions)
Dna blood mini kit, by Qiagen (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Ultrapower, by BioTeke (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
4 protocols using ingenius
1
Watermelon Virus Identification via PCR
2
TLR4 Polymorphism Identification via PCR-RFLP
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The extraction step was performed with a DNA blood mini kit (Qiagen) using a peripheral
blood sample. The PCR-RFLP method was used to identify polymorphisms. Asp299Gly
polymorphism was present at the Ncol enzyme restriction site. Thr399lle polymorphism was
present at the Hinf 1 restriction site. The primers are listed inTable 1 (7 (link)). The
amplification step was performed with the GeneAmp PCR system 9700 (Applied
Biosystems).
The PCR master mix comprised 1 mL DNA solution, GeneAmp Gold buffer (15 mmol/L Tris HCl;
pH 8.0), 50 mmol KCl, 1.5 mmol MgCl2, 50 mmol/L each of dGTP, dATP, dTTP, and
dCTP (Promega), 25 pmol forward and reverse primers, and 1.0 U AmpliTaq Gold polymerase
(Applied Biosystems). The PCR reaction was performed at a 25-µL volume and involved
denaturing at 95°C for 10 minutes, annealing at 59°C for 45 minutes, extension
at 72°C for 45 minutes, and a final extension at 72°C for 7 minutes. The PCR gel
image was visualized in 2% ethidium bromide-containing agar (117 mA, 102 mV) (OWL
separation systems) under UV light (InGenius; Syngene). The PCR products then underwent
restriction by restriction enzymes (New England Biolabs) to a final volume of 10 µL
at 37°C overnight incubation. The restriction took place in a 1-mL solution with 2
and 4 U of Acil enzyme. The restriction products were visualized in 3% ethidium
bromide-containing gel under UV light (100 mV, 60 minutes).
blood sample. The PCR-RFLP method was used to identify polymorphisms. Asp299Gly
polymorphism was present at the Ncol enzyme restriction site. Thr399lle polymorphism was
present at the Hinf 1 restriction site. The primers are listed in
amplification step was performed with the GeneAmp PCR system 9700 (Applied
Biosystems).
The PCR master mix comprised 1 mL DNA solution, GeneAmp Gold buffer (15 mmol/L Tris HCl;
pH 8.0), 50 mmol KCl, 1.5 mmol MgCl2, 50 mmol/L each of dGTP, dATP, dTTP, and
dCTP (Promega), 25 pmol forward and reverse primers, and 1.0 U AmpliTaq Gold polymerase
(Applied Biosystems). The PCR reaction was performed at a 25-µL volume and involved
denaturing at 95°C for 10 minutes, annealing at 59°C for 45 minutes, extension
at 72°C for 45 minutes, and a final extension at 72°C for 7 minutes. The PCR gel
image was visualized in 2% ethidium bromide-containing agar (117 mA, 102 mV) (OWL
separation systems) under UV light (InGenius; Syngene). The PCR products then underwent
restriction by restriction enzymes (New England Biolabs) to a final volume of 10 µL
at 37°C overnight incubation. The restriction took place in a 1-mL solution with 2
and 4 U of Acil enzyme. The restriction products were visualized in 3% ethidium
bromide-containing gel under UV light (100 mV, 60 minutes).
3
PCR-based Detection of Trypanozoon
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DNA extractions were performed using commercial DNA extraction kits (Qiagen®, QIAamp® DNA Blood Mini Kit, Maryland, USA), according to the manufacturer’s instructions. PCR was conducted using TBR1/2 primers to amplify a 164 bp product from a highly repeated sequence of mini-chromosome satellite DNA for the detection of Trypanozoon (Table-1 ), following a protocol previously described [21 (link),22 (link)]. Briefly, 18 μL PCR mixtures were prepared using PCR buffer, 50 mM MgCl2, 10 mM dNTPs mixed, 10 μM of each primer, 0.5 Unit of taq DNA polymerase (Invitrogen™, Life Technologies, California, USA), and 2 μL of DNA sample. Trypanosoma evansi DNA was extracted from the blood of an experimentally infected mouse for the positive control, and distilled water was used as the negative control. Initial denaturation was 94°C 1 min followed by 30 cycles of denaturation at 94°C 30 s, annealing at 60°C 1 min, extension at 72°C 30 s, and an extra final extension at 72°C 2 min. PCR product was stained using non-toxic nucleic acid stains (Ultrapower®, BioTeke, Beijing, China) and migrated through 1.5% agarose gel (Biotech™, Bio Basic Canada Inc., Ontario, Canada) in a submarine electrophoresis system (Advance Mupid-exU TM, Takara Bio Inc., California, USA). PCR products were observed under a UV transilluminator (InGenius™, Syngene Bio Imaging®, Labnet International Inc., Woodbridge, USA).
4
Yeast Two-Hybrid Screening Protocol
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Yeast two-hybrid assays were performed by co-transforming GBD and GAD (Gal4-activating domain) plasmids (in the pGBKT7 and pGADT7 vectors) in various combinations in the PJ69-4A budding yeast strain (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4 gal80 LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ). Transformants were screened for interaction by spotting 5-fold dilutions on minimal plates lacking tryptophan and leucine for selection of GBD and GAD plasmids, respectively. Interactions were assessed by quantification of the expression of the HIS3 and ADE2 markers by plating on minimal medium lacking, in addition, histidine or adenine. The stringency of the HIS3 reporter was also increased by the addition of 3 mM 3-aminotriazole. The plates were then incubated at 30 °C for 3 (SC-leu-trp) or 4 (selective plates) days, imaged with a Syngene InGenius instrument and processed with Photoshop.
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