MMP-2 and MMP-9 activity was assessed via gelatin zymography. 10 μl of BAL supernatant was diluted 1:1 in Tris-Gly SDS zymogram samples buffer (Invitrogen), and incubated at 21 °C for 10 min. 15 μl of sample (or standard) was loaded onto zymogram precast gelatin gels, 10 % Tris-Glycine gel with 0.1 % gelatin substrate (Invitrogen) and subjected to electrophoresis at constant voltage of 125 V for 100 min. Gels were incubated at RT for 60 min in 100 ml of zymogram renaturing buffer, 2.5 % Triton X-100 (Invitrogen), to recover enzymatic activity. Gels were then incubated in zymogram developing buffer, 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2 (Invitrogen) for 1 hour prior to overnight incubation at 37 °C. Gels were then stained with coomassie blue-stain (Coomassie blue 0.1 % (w/v) in H2O 50 % (v/v), methanol 40 % (v/v) and acetic acid 10 % (v/v)) for 60 min before destaining in the same solution without the dye for 30 min. Bands were then visualized by image analysis (Gel Doc, Syngene, Cambridge, UK) and densitometry determined using Image J software. Band densities were standardized to the mean density of two control samples, per gel, to allow comparisons between multiple gels. All gels were run in parallel.
Gel doc
Manufactured by Syngene
Sourced in United Kingdom, United States
The Gel Doc is a lab equipment used for the analysis and documentation of nucleic acid and protein electrophoresis gels. It captures and digitizes gel images for further processing and analysis.
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Lab products found in correlation
Tris glycine gel, by Thermo Fisher Scientific (2 mentions)
Cacl2, by Thermo Fisher Scientific (2 mentions)
Tris gly sds zymogram samples buffer, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Lourmat, by Vilber (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Dnazol, by Thermo Fisher Scientific (1 mentions)
Uv spectrophotometer, by Shimadzu (1 mentions)
Gelcompar software, by bioMérieux (1 mentions)
Dna ladder, by Thermo Fisher Scientific (1 mentions)
15 protocols using gel doc
1
Quantifying MMP-2 and MMP-9 Activity
2
Specific Detection of Pigeonpea Sterility Mosaic Virus
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SMV-1 and SMV-2 oligonucleotide primers (known for the amplification of a 321-bp product) were developed from partial nucleocapsid sequence (764 bp; AJ439561) of PPSMV-P8 to determine the specificity of these primers to PPSMV-I and PPSMV-II. About 1 µg of total RNA from SMD affected Mg-1, Mg-2, Bng-1, Bng-2, Bng-3, CO-5 CO-6 and pigeonpea cv. ICP8863 and mite inoculated ICP8863 (PPSMV-P sub-isolate Chevella) leaf tissues were used as template for the assay. The protocols for RT reaction, first strand cDNA synthesis and polymerase chain reaction of the primers were followed as described8 (link). Total RNA from the non-symptomatic pigeonpea cv. ICP8863 and unknown cultivar (Bengaluru) from the three locations were used as control. RT-PCR amplicons were fractionated on 1% agarose gel containing ethidium bromide and visualized in a gel doc (Syngene, UK).
3
Genomic-SSR Genotyping of Indian Mustard
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A set of 227 genomic-SSR markers covering all the linkage groups of Indian mustard as reported by Singh et al. (2021) were used in present study for genotyping. The mastermix was prepared in a 2.0 ml centrifuge tube in 25 μl volume by taking 50 ng genomic-DNA, 1XPCR buffer, 2.0 mM Mgcl2, 0.2 mM of each dNTP, 1.0 U Taq DNA polymerase (GCC Biotech, India) and 400 nM primers using Verity 96-w PCR machine. In thermal cycler, initial denaturation cycle comprised of 94 °C temperature for 5 min which was followed by 45 cycles at 94 °C for 30 s, 50–60 °C (depending on the annealing temperature of the SSR primers) for 30 s, 45 s of extension at 72 °C and in the last, primer extension step at 72 °C for 7 min. PCR amplicons containing 5 μl loading dye were resolved in a 3.5% Super Fine Resolution (SFR) agarose (Amresco, USA). Further, gel pictures were taken in a Gel Documentation unit (Syngene Gel Doc, UK).
4
Mapping DNA Breakpoints in ABL Gene
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The PCR products were analysed on 1% agarose gel. The agarose gel electrophoresis was performed at 90 V for 1 h and 30 min. The agarose gel was briefly stained with ethidium bromide (0.5 μg/ml) and destained with distilled water. This was followed by visualisation of the gel on an ultraviolet (UV) transilluminator (Vilber Lourmat). The gel image was captured and analysed using a gel documentation (gel doc) and image analysis system (Syngene). The IPCR bands representing cleaved DNA fragments of the ABL gene were purified by using QIAquick Gel Extraction Kit (QIAGEN) according to the manufacturer’s protocol and sequenced. By blasting the human genome database (Genomic BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi ), the sequencing data obtained was annotated. To identify the breakpoints of the cleaved fragments, the sequencing data was analysed and aligned with the published ABL gene sequence [Ensembl:ENSG00000097007] by using Seqman DNASTAR software (Lasergene, USA). The positions of DNA breaks identified were compared with the location of the MAR/SAR sequence isolated experimentally in the previous study [77 (link)] and the MRS identified in the present study. A genomic map was constructed to depict the positions of the detected DNA breaks relative to the location of the MAR/SAR.
5
Quantifying MMP-2 and MMP-9 Activity
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MMP-2 and MMP-9 activity was assessed via gelatin zymography. 10 μl of BAL supernatant was diluted 1:1 in Tris-Gly SDS zymogram samples buffer (Invitrogen), and incubated at 21 °C for 10 min. 15 μl of sample (or standard) was loaded onto zymogram precast gelatin gels, 10 % Tris-Glycine gel with 0.1 % gelatin substrate (Invitrogen) and subjected to electrophoresis at constant voltage of 125 V for 100 min. Gels were incubated at RT for 60 min in 100 ml of zymogram renaturing buffer, 2.5 % Triton X-100 (Invitrogen), to recover enzymatic activity. Gels were then incubated in zymogram developing buffer, 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2 (Invitrogen) for 1 hour prior to overnight incubation at 37 °C. Gels were then stained with coomassie blue-stain (Coomassie blue 0.1 % (w/v) in H2O 50 % (v/v), methanol 40 % (v/v) and acetic acid 10 % (v/v)) for 60 min before destaining in the same solution without the dye for 30 min. Bands were then visualized by image analysis (Gel Doc, Syngene, Cambridge, UK) and densitometry determined using Image J software. Band densities were standardized to the mean density of two control samples, per gel, to allow comparisons between multiple gels. All gels were run in parallel.
6
DNA Extraction and Analysis in HaCaT Cells
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HaCaT cells (1.6 × 106 cells/dish) were cultured in a 6 cm cell culture dish for 24 h. DSE was added to the culture 24 h before NB-UVB exposure (pretreatment with DSE) or after NB-UVB exposure (posttreatment with DSE) and further incubated for 24 h. Then, the cells were harvested and DNA was extracted by using DNAzol (Invitrogen) according to the manufacturer's protocol. The genomic DNA was measured for A260 and A280 by using a UV spectrophotometer (Shimadzu, Japan). Five micrograms of DNA was loaded into a 1.8% (w/v) agarose gel and photographed by using Gel Doc (SynGene).
7
PCR and Gel Electrophoresis for ITS Region
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To amplify the ITS region, we used primers forward: 5′-GAAGTCGTAACAAGG-3′ and reverse: 5′-CAAGGCATCCACCGT-3′ as previously described [17] (link). The PCR was performed with 20-μL reaction volumes comprising 10 μL Master mix (Ampliqon, Odense, Denmark), 0.5 μL forward primer 10 pmol (Bioneer, Daejeon, Korea), 0.5 μL reverse primer 10 pmol (Bioneer, Daejeon, Korea), 8.5 μL distilled water and 50 ng bacterial DNA.
The PCR was performed in a thermocycler (PEQLAB, Erlangen, Germany) with an initial denaturation at 95°C for 5 min; and 25 cycles, including denaturation steps at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 5 min. Electrophoresis of the PCR product was performed in an 8% polyacrylamide gel. The gel was stained using 1% silver nitrate and detection used Gel Doc (GVM20 model syngene, Cambridge, UK). The digital image was stored electronically as a TIFF image and analysed with GelCompar software (Applied Maths, Sint-Martens-Latem, Belgium) by using the Dice correlation coefficient and the UPGMA method.
The PCR was performed in a thermocycler (PEQLAB, Erlangen, Germany) with an initial denaturation at 95°C for 5 min; and 25 cycles, including denaturation steps at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 5 min. Electrophoresis of the PCR product was performed in an 8% polyacrylamide gel. The gel was stained using 1% silver nitrate and detection used Gel Doc (GVM20 model syngene, Cambridge, UK). The digital image was stored electronically as a TIFF image and analysed with GelCompar software (Applied Maths, Sint-Martens-Latem, Belgium) by using the Dice correlation coefficient and the UPGMA method.
8
Gelatin Zymography of Aortic Supernatants
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Supernatants collected from rat aortic ring wells were run on 7.5 % gel made with 0.1 % gelatin concentration. The supernatant was mixed with reducing agent-free loading dye. Electrophoresis was carried out until tracking dye was rundown, and gel was washed in washing buffer and kept overnight in reconstitution buffer in a CO2 incubator. It was stained with Coomassie brilliant blue dye for 20 min and then was destained with 7 % glacial acetic acid. The zymogram was scanned using a GEL DOC (Syngene).
9
Optimizing Blood Lysis for PCR
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Initially, the GAPDH gene of the host was amplified to acquire baseline data on the effective concentration of NaOH to be utilized in the preparation of blood lysate (mainly dogs). Accordingly, 10 µl of apparently healthy animal blood was arbitrarily included in 0.6 ml tubes containing 200 µl of NaOH solution of varying concentrations starting with 5 mM to 80 mM with a difference of 5 mM. One test tube containing 200 µl of nuclease-free water was also kept as a negative control. The mixture was vortexed for a few seconds and incubated in a water bath maintained at 90 °C ± 5 °C for 10 min. After incubation, the mixture was vortexed for a brief period and centrifuged at 6000×g for 5 min. Now, this blood lysate (4 µl) was used as the template in place of the purified DNA in a 25 µl PCR reaction as mentioned in the previous section. The blank 25 mM NaOH was included in PCR as a negative control. The PCR reaction was performed at optimized thermocyclic conditions and 10 µl of PCR product along with DNA ladder (Thermo Scientific, Vilnius, Lithuania) was electrophoresed, visualized and documented in Gel Doc (Syngene, USA).
10
Molecular Detection of Haemoparasites in Animals
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The laboratory-standardized PCR primers ( Table 1 ) were used in the present study for the identification of haemoparasitic infections in animals through PCR assay. The standard 25 µl PCR reaction was set up in 200 µl PCR tubes as mentioned in the previous section. The haemoparasite positive DNA was used as positive control while without template DNA was used as negative control. The reaction mixture was incubated in a Gradient thermal cycler (Applied Bio System, USA) at standard PCR conditions as Initial denaturation (95 °C for 5 min), 35 cycles of denaturation (96 °C for 15 s), annealing (as per the primers, see Table 1 ) and extension (as per the primers, see Table 1 ) and final extension (72 °C for 5 min). After the reaction, 10 µl of PCR products were loaded in 1.2% agarose gel containing ethidium bromide (0.5 µg/ml) and electrophoresis was done at 100 V for 40 min. The DNA on the gel was visualized and documented using Gel Doc (Syngene, USA).
Table 1. Details of PCR primers used for identification of various haemoprotozoans of animals through PCR assay.
Table 1. Details of PCR primers used for identification of various haemoprotozoans of animals through PCR assay.
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