The expression plasmids pGE-hRID(2m)-6xhis-EGFP (hRID(2m)-EGFP) and pGE-LysRS-6xhis-SPARC (RS-SPARC), were transformed into the BL21(DE3)pLysE and BL21(DE3)pLysS E. coli strains, respectively. A single colony was inoculated into 3 mL Luria-Bertani (LB) medium containing 50 µg/mL ampicillin and 34 µg/mL chloramphenicol, then cultured overnight at 37 °C. Approximately 0.5 mL of the overnight cultured cells were transferred to 20 mL of fresh LB medium with the same concentration of antibiotics, then cultured at 37 °C until an optical density (OD)600 of 0.5 was reached. The expression of hRID(2m)-EGFP or RS-SPARC was then induced by adding 1 mM Isopropyl beat-D-1-thiogalactopyranoside (IPTG) followed by incubation for 3 h at 37 °C or 5 h at 27 °C. Approximately 10 mL of cultured cells were harvested and suspended in 0.3 mL of the mild lysis buffer B-PER (Thermo Scientific, 90078, Rockford, IL, USA). The suspended cells were sonicated or incubated at room temperature, and separated into total, soluble, and insoluble fractions by centrifugation at 15,000× g for 12 min at 4 °C. The expression of target proteins was analyzed by SDS-PAGE. The fluorescent protein bands and Coomassie Blue stained protein gels were imaged using a gel documentation system (InGenius3, SYNGENE, Frederick, MD, USA).
Ingenius3
Manufactured by Syngene
Sourced in United States, United Kingdom
InGenius3 is a lab equipment product designed for DNA and RNA analysis. It performs nucleic acid separation and purification using centrifugation technology. The core function of InGenius3 is to isolate and extract genetic material from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
B per, by Thermo Fisher Scientific (1 mentions)
Dnase 1 amplification grade, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (1 mentions)
Gelred, by Biotium (1 mentions)
Genesys image acquisition software, by Syngene (1 mentions)
Mastercycler, by Eppendorf (1 mentions)
Gotaq pcr master mix, by Promega (1 mentions)
Phusion green hot start 2 high fidelity pcr master mix, by Thermo Fisher Scientific (1 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Taq polymerase buffer, by Thermo Fisher Scientific (1 mentions)
Loading dye, by Promega (1 mentions)
Dnaeasy plant mini kit, by Qiagen (1 mentions)
7 protocols using ingenius3
1
Recombinant Protein Expression and Purification
2
Genomic DNA extraction from Fusarium
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The genomic DNAs of isolates were extracted using the CTAB method (Li et al., 2013 ). The 10 most virulent Fusarium isolates were incubated on PDA plates at 27 ± 2°C under the dark for 6–7 days. An approximately 1 cm2 of fungal mycelium was transferred from culture plates to a sterile 2 ml Eppendorf tube. Mycelium was macerated using a tissue homogenizer and 500 μl CTAB buffer, followed by incubation of the Eppendorf tube at 60°C for 1 h in a hot water bath (Cole-Palmer India Pvt. Ltd. Mumbai) with a gentle shaking at 10 min interval. The composition of the buffer was (2.5% CTAB, 4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, 0.2% β- Mercaptoethanol; pH 8.0). All components for the buffer preparation were procured from (Hi-media, India). The concentration and purity of DNA were determined using Nanodrop (Nanodrop™ ThermoFisher™ Scientific, Mumbai, India) and recording its absorbance at (260/280 nm) and the quality of DNA through agarose gel electrophoresis (0.8%) (w/v) (iGene Labserve, Delhi, India). The gel was observed under the Gel documentation system (Syngene®InGenius3, Frederick, USA). The final volume of DNA was made to 50 ng.
3
Quantitative Gene Expression Analysis of Pancreatic Cells
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Total RNA was extracted using TRIzol® Reagent (Thermo Fisher®, Waltman, USA) and samples were treated with DNase I, Amplification Grade (Thermo Fisher®, USA), before cDNA synthesis using SuperScript® III First-Strand Synthesis SuperMix (Thermo Fisher®, USA). RT-PCR was subsequently performed using an Eppendorf® Mastercycler (Eppendorf™, Hamburg, Germany) to determine the relative expression levels of selected pancreatic genes using GoTaq PCR Master Mix (Promega®, Madison, USA) and the previously published oligonucleotide sequences and optimised PCR protocols (Supplementary Table 1 ) [4 (link)]. PCR products were imaged after electrophoresis on a 1% w/v agarose gel stained with 10000x GelRed™ (Biotium®, Fremont, USA) (1 : 100000) on the InGenius3 (Syngene®, Frederick, USA) UV transilluminator using the GeneSys image acquisition software (Syngene®).
4
Extracellular Vesicle RNA Profiling
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The sEVs RNA (200 ng) was used for cDNA synthesis with the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, United States) following the manufacturer’s instruction. The cDNA was used as a template for PCR, which was performed with Phusion Green Hotstart II High-Fidelity PCR Mastermix (Thermo Fisher Scientific, Waltham, MA, United States). The following cycling conditions were applied: Initial denaturation at 98°C for 2 min, 30 cycles of denaturation 98°C/10 s, 60°C/30 s, 72°C/45 s, and final extension at 72°C for 5 min. The PCR amplicons were separated on 1% agarose gel and the images were captured in InGenius3 (Syngene, Cambridge, UK).
5
Genotyping of MTHFR C677T Polymorphism
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To genotype the SNP of the MTHFR C677T (rs1801133) gene, Tetra-ARMS PCR was used. Three types of genotypes (C/C, C/T, and T/T) were found for the MTHFR C677T (rs1801133) gene. The base pair size used for Tetra ARMS PCR was 86 bp and 146 bp. For the amplification of the MTHFR C677T (rs1801133) gene, two forward primers and two reverse primers were used (Table 1 ). At the start of the reaction, two non-allele specific primers were amplified by the outer primers which produced outer fragments that served as a template for the attachment and elongation of inner primers.
A Thermocycler Master Cycler Gradient was used to perform PCR. PCR was carried out in a 20 µL container with 1 µL of DNA sample, 10 µL Master Mix, 0.1–0.5 mM of each primer, and 8 µL of DD-H2O. The initial denaturation temperature was 95 °C for 5 min, further followed by 40 cycles of denaturation at 94 °C for 30 s. Annealing was performed at 52 °C for 75 s, followed by extension at 72 °C for 40 s. The final extension temperature was again 72 °C for about 7 min. After the completion of the PCR reaction, 20 μL of PCR resultant product was placed in the well with 2% agarose gel, dyed with ethidium bromide, soaked in TAE buffer, and let to run in an electric field. The gel was then examined under UV light using a gel documentation system (InGenius3, Syngene, UK).
A Thermocycler Master Cycler Gradient was used to perform PCR. PCR was carried out in a 20 µL container with 1 µL of DNA sample, 10 µL Master Mix, 0.1–0.5 mM of each primer, and 8 µL of DD-H2O. The initial denaturation temperature was 95 °C for 5 min, further followed by 40 cycles of denaturation at 94 °C for 30 s. Annealing was performed at 52 °C for 75 s, followed by extension at 72 °C for 40 s. The final extension temperature was again 72 °C for about 7 min. After the completion of the PCR reaction, 20 μL of PCR resultant product was placed in the well with 2% agarose gel, dyed with ethidium bromide, soaked in TAE buffer, and let to run in an electric field. The gel was then examined under UV light using a gel documentation system (InGenius3, Syngene, UK).
6
PER3 VNTR Genotyping Protocol
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DNA was isolated from peripheral blood using standard salting-out procedure [11] . PER3 VNTR variants were determined with PCR technique with previously described primer sequences [9] . PER3 PCR reaction was carried out in a 25 µl total volume containing ~100 ng genomic DNA, 0.5 µM of each primer, 0.2 mM of each dNTP, 2 mM MgCl2, 1x Taq polymerase buffer, and 1.5 U Taq polymerase (Thermo Fisher Scientific, Inc.). Thermal cycler condition was as follows: 35 cycles of denaturation for 40 sec at 94 o C, annealing for 45 sec at 55 o C and extension for 45 sec at 70 o C. A predenaturation step for 6 min at 94 o C and a final extension step for 12 min at 70 o C were included. Post PCR products were loaded on 2.5% ultra-pure agarose gel, stained with ethidium bromide, electrophoresed and analyzed with gel documentation system (SYNGENE Ingenius 3, England) to enable to the analysis of ins/del polymorphism to distinguish the 5-repeats allele (401 bp) from the 4-repeats allele (347 bp). Heterozygotes (4/5) resulted with both bands.
7
Genomic DNA Extraction from Leaves
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Genomic DNA (gDNA) was isolated from young unfolding leaf tissues. About 1 g of fresh leaf tissue was grounded into a fine powder in prechilled mortar and genomic DNA from individual accession was extracted using, DNAeasy Plant Mini Kit (QIAGEN, USA). The DNA extraction was performed in accordance with the manufacturer's instruction. DNA concentration were determined comparatively by electrophoresis at current of 100 amps, 80 volts, for 40 minutes using agarose gel (0.8 %) electrophoresis, by applying 5 μl gDNA loaded after mixing with 3 μl 6X loading dye (Promega, USA) to check the quality of the DNA by comparing the intensity of the bands with a 1kb standard (Thermo Scientific, USA). The gel was visualized under a UV transilluminator and the gel was imaged with a gel documentation system (Ingenius-3, Syngene, USA) to confirm the quality of the genomic DNA.
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