Doyle and Doyle [93 ] method was used to isolate and purify DNA from fresh leaves (approximately 0.25 g) of the donor plant growing in soil in the botanical garden and ex-vitro-established micropropagated plant. Using a Nanodrop spectrophotometer (Nanodrop 2000, Thermo Scientific, Waltham, MA, USA), the purity and concentration of DNA was determined. Ten ISSR primers (UBC, Vancouver, BC, Canada) were used to check the genetic fidelity of micropropagated plant. PCR reactions were performed in a 25 μL reaction volume containing 1.5 µL genomic DNA (50 ng/μL), 1.5 µL primer, 12.5 µL of PCR master mix (GoTaq® Green Master Mix, 2X, Promega, Madison, WI, USA), and 9.5 µL ultrapure (Milli-Q) water. PCR reaction was carried out in a Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the following conditions: initial denaturation for 5 min at 94 °C, 34 cycles of denaturation step at 94 °C (45 s), primer annealing 46–55 °C (30 s), extension at 72 °C (90 s), and a final extension step for 5 min at 72 °C. To avoid false results and to ensure that the ISSR markers performed the same way each time, all the experiments were repeated three times. PCR products were photographed by a gel documentation system (G: BOX F3, Syngene, Cambridge, UK) after being separated by electrophoresis on a 1.5% (w/v) agarose gel in 1X Tris acetic acid EDTA (TAE) buffer at 75 V.
G box f3
Manufactured by Syngene
Sourced in United Kingdom, United States
The G:BOX F3 is a compact, versatile gel documentation system designed for imaging a variety of samples, including DNA, RNA, and protein gels. It features a high-resolution camera, adjustable UV and white light illumination, and intuitive software for capturing and analyzing images.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Ethidium bromide, by Merck Group (2 mentions)
Tris borate edta, by Merck Group (2 mentions)
Pvdf membrane, by Roche (2 mentions)
Instantblue, by Abcam (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Thermal cycler, by Bio-Rad (1 mentions)
Sequencing service, by Macrogen (1 mentions)
Alexa fluor 568 anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Genesys software, by Syngene (1 mentions)
A phosphatase inhibitor, by Solarbio (1 mentions)
Anti ptrf, by Abcam (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti cd63, by Santa Cruz Biotechnology (1 mentions)
Bca kit, by Beyotime (1 mentions)
31 protocols using g box f3
1
Genetic Fidelity Verification of Micropropagated Plants
2
Molecular Sexing and Parasites Screening in Birds
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Genomic DNA was extracted using the salting-out procedure (Aljanabi and Martinez, 1997 (link)). The sex of birds was determined by using a molecular method (Fridolfsson and Ellegren, 1999 ). Polymerase Chain Reaction (PCR) products were run in 1% agarose gels, stained with Syber Safe®, and visualized using the system for the documentation and analysis of fluorescently stained gels GBOX F3 (Syngene, MD, USA). Birds were sexed as females (heterogametic: WZ) and males (homogametic: ZZ).
Screening for parasites were performed with a parasite genus-specific primers in a nested-PCR protocol that amplifies a fragment of 480 bp (excluding PCR primers) of the mitochondrial cytochrome b (cyt b) gene of haemosporidian parasites (Hellgren et al., 2004 (link)). Two positive controls for parasites and two negative controls (ddH2O) for each 48 samples were included, no contamination was detected. We screened each sampled at least twice, to avoid false negatives. Positive PCR products were purified and sequenced using the Macrogen sequencing service (Macrogen Inc., South Korea).
Screening for parasites were performed with a parasite genus-specific primers in a nested-PCR protocol that amplifies a fragment of 480 bp (excluding PCR primers) of the mitochondrial cytochrome b (cyt b) gene of haemosporidian parasites (Hellgren et al., 2004 (link)). Two positive controls for parasites and two negative controls (ddH2O) for each 48 samples were included, no contamination was detected. We screened each sampled at least twice, to avoid false negatives. Positive PCR products were purified and sequenced using the Macrogen sequencing service (Macrogen Inc., South Korea).
3
Western Blot Analysis of Signaling Proteins
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Total cell lysates were prepared by harvesting cells in Laemmli SDS reducing buffer. Protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA), resolved on an 8% to 10% polyacrylamide gel, and transferred to a polyvinylidine fluoride membrane. The following antibodies are from Cell Signaling (Beverly, MA, USA): glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118), p-JNK (4668), JNK (9258), p-p38 (4511), p38 (8690), p-NFkappa (3031), NFkappa (8242), p-ERK1/2 (4377), ERK 1/2(4695), p-RIP2 (4364). RICK (RIP2) antibody (sc-136059) was from Santa Cruz Biotech (Dallas, TX, USA). Focal adhesion kinase (FAK) (ab40794) and p-FAK (ab4803) antibodies were from Abcam (Cambridge, MA). Detection of peroxidase activity from HRP-conjugated antibodies was done with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL, USA). Images were captured with the G:BOX F3 with GeneSys software (SynGene, Frederick, MD). Alexa Fluor 568 anti-rabbit secondary antibody (Invitrogen) was used for immunofluorescence and images were taken at 100× with Zeiss Axiovert 200M (Thornwood, NY, USA).
4
Western Blot Analysis of Cell and Exosome Samples
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Western blot analysis of cells, samples and exosome lysates was performed according to the manufacturer's instructions and a previously described protocol 28 (link). Briefly, cells, samples and exosomes were lysed with RIPA buffer (Solarbio, Beijing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor (Solarbio), and lysates were incubated on ice for 30 min to ensure membrane disruption. Protein concentrations were determined using a bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China) following the manufacturer's recommendations, and electrophoresis was performed using 10% acrylamide gels. The following primary antibodies were used: anti-EGFRvIII, anti-EGFR, and anti-p-AKT Ser473 (Cell Signaling Technology (CST), Boston, MA, USA, 1:1,000 dilution), anti-PTRF (Abcam, Cambridge, MA, USA, 1:1,000 dilution), anti-CD63 (Santa Cruz Biotechnology, CA, USA, 1:500 dilution) and anti-GAPDH (Millipore, Billerica, MA, USA, 1:2,000 dilution). Anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Promega (Madison, WI, USA, 1:10,000 dilution). Immunoblots were developed using G:BOX F3 (Syngene, Cambridge, UK).
5
Oligomycin-Induced Colony Formation Assay
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The colony formation assay was performed as described previously.[13 ] Briefly, cells were seeded in 60 mm dishes at a density of 2 × 103 cells per well. Three days later, the cells were treated with oligomycin at a final concentration of 2 µm for 10 days. Medium with or without the inhibitor was replaced every 3 days. When the colonies formed by control cells were ≈ 50 cells per colony, the cells were fixed with methyl alcohol for 15 min; and then, stained with a 0.5% crystal violet solution for 15 min. The colonies were counted by a gel documentation system (G:BOX F3, Syngene, USA) and analyzed. Experiments were performed in triplicate and the results are presented as the mean ± SD.
6
Western Blot Analysis of Mitochondrial Proteins
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Protein lysates were prepared from flash-frozen cell pellets following sonication. Total protein concentration was quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher). Total protein per sample (30 µg) was run in NuPAGE 10% Bis-Tris 1.0–1.5 mm mini protein gels (Invitrogen) and then transferred onto polyvinylidene difluoride membranes (Invitrogen) using an XCell SureLock Mini-Cell (Invitrogen). Blots were blocked for 1 h at room temperature with 5% non-fat dry milk dissolved in Tris buffered saline with Tween. Primary antibodies used were mouse monoclonal anti-MT-CO1 (1D6E1A8, no. ab14705, Abcam, 1:1,000 dilution), mouse monoclonal anti-NDUFB8 (20E9DH10C12, no. ab110242, Abcam, 1:1,000 dilution) and mouse monoclonal anti-alpha-tubulin (DM1A, Loading Control, no. ab7291, Abcam, 1:20,000 dilution). The secondary antibody was goat anti-mouse IgG H&L (horseradish peroxidase, Abcam, 1:5,000 dilution). Primary antibodies were incubated overnight at 4 °C and the secondary antibody was incubated for 1 h at room temperature. Membranes were developed with Amersham ECL Prime Western Blotting Detection Reagent (Cytiva) and imaged using G:Box F3 (Syngene).
7
Biofilm-Associated Genes in Staphylococci
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The following biofilm-associated genes: icaA, icaD, eno, bap among all staphylococci, and in addition bhp, aap, fbe, embP, atlE among CoNS were amplified by PCR using specific primers (Table S1 ). The PCR products were visualized by electrophoresis on 1.5% agarose gels in 1×TBE (Tris-borate-EDTA) buffer stained by 0.5 μg/mL of ethidium bromide (0.5 mg/mL; Sigma-Aldrich Corp., St. Louis, MO, USA) and visualized using the system for the documentation and analysis of fluorescently stained gels G-BOX F3 (Syngene, Cambridge, UK).
8
Immunoblotting Analysis of Plant Proteins
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Plant homogenates containing 30 µg of TSP were mixed with non‐reducing NuPAGE LDS sample buffer and boiled for 10 min. Proteins were separated on a 4%–12% Bis‐Tris NuPAGE gel (Life Technologies) with MOPS buffer and transferred to a nitro‐cellulose membrane for immunoblotting. The membrane was blocked with 5% w/v non‐fat dried milk (NFDM) in Tris‐buffered saline supplemented with 0.1% v/v Tween20 (TBS‐T) before being probed with either peroxidase‐conjugated polyclonal sheep anti‐human kappa LC antibody (1 in 10,000), peroxidase‐conjugated sheep anti‐human IgG gamma chain (1 in 5000), peroxidase‐conjugated monoclonal anti‐polyhistidine antibody (1 in 2500; all from Sigma) or rabbit anti‐GRFT (1 in 2000; gift from Barry O'Keefe). Anti‐GRFT blots were subsequently incubated with peroxidase‐conjugated sheep anti‐rabbit IgG antiserum (1 in 2000; Sigma). Protein detection was done using the ECL Prime system (Thermo Fisher) and were visualized using G:Box F3 (Syngene, UK) or Amersham Hyperfilm ECL (GE Healthcare).
9
Collagen Protein Extraction and Analysis
10
Protein Expression Analysis by SDS-PAGE and Immunoblotting
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SDS/PAGE and immunoblotting assays were performed as described previously (Hosur et al., 2014 (link)). Briefly, MEFs grown in six-well dishes were lysed with RIPA buffer (Cell Signaling Technology) and protein concentrations were determined using a Qubit Fluorometer (Life Technologies). After loading 50 μg of protein onto 4-20% (wt/vol) precast gels (Lonza), proteins were transferred to a PVDF membrane, before blocking with 5% milk for 1 h at room temperature (RT). Membranes were then exposed to EGFR (#2232; 1:1000), phospho-ERK1/2 (#4370; 1:1000), phospho-AKT (#4060; 1:1000), phospho-S6 (#4858,; 1:1000 dilution) or actin (#4970; 1:1000) antibodies (Cell Signaling Technology) overnight at 4°C. Subsequently, membranes were washed in TBST for 2 h, exposed to secondary antibodies (Santa Cruz Biotechnology; 1:10,000) for 1 h at RT, and washed for 2 h in TBST. Membranes were then exposed to Luminol reagent (Santa Cruz Biotechnology) for 3 min and visualized using a gel documentation system (G:BOX F3, Syngene, Frederick, USA).
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