Denaturing gradient gel electrophoresis was performed using a Dcode Mutation Detection System (BioRad Laboratories Ltd., Hertfordshire, UK). PCR products were electrophoresed with 0.5x TAE buffer (1x TAE buffer is 0.04 M Tris base, 0.02 M sodium acetate, and 10 mM EDTA [pH 7.4]) on 8% acrylamide gel containing a 25 to 50% denaturating gradient of formamide and urea. DGGE was conducted at 60°C for 5 h at 200 V. The gel was then treated with SYBR Green I Nucleic acid gel stain (Cambrex Bio Science Rockland, USA), photographed and analyzed for DGGE band profile using an UV gel documentation system (BioRad Laboratories Inc., Hercules, CA, USA).
Dcode mutation detection system
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
The DCode Mutation Detection System is a laboratory equipment designed for the analysis of DNA mutations. It utilizes denaturing gradient gel electrophoresis (DGGE) technology to separate and detect DNA fragments based on their sequence differences. The system provides a reliable and efficient method for screening and identifying genetic variations.
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Lab products found in correlation
Sybr green 1 nucleic acid gel stain, by Cambrex (3 mentions)
Uv gel documentation system, by Bio-Rad (3 mentions)
G box f3 system, by Syngene (2 mentions)
Model 475, by Bio-Rad (1 mentions)
Genematrix soil dna purification kit, by EURx (1 mentions)
Deionized formamide, by Sangon (1 mentions)
Quantity one 1 d software, by Bio-Rad (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
10 protocols using dcode mutation detection system
1
Denaturing Gradient Gel Electrophoresis
2
Microbial Diversity Analysis via DGGE
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The experiments were performed as described previously (42 (link)). Briefly, HDA1 and HDA2-GC primers were used to assess microbial diversity in each sample. PCR products were separated in 8% polyacrylamide gels with a range of 30–50% denaturing gradient (100% denaturant consisted of 7 M urea and 40% deionized formamide) cast with Bio-Rad’s Model 475 gradient delivery system (BioRad, Hercules, CA, USA). The electrophoresis was performed in TAE 0.5× buffer for 5 h at a constant electric current of 125 mA and a temperature of 60°C with the DCode Mutation Detection System (Bio-Rad, Hercules, CA, USA). Clustering analysis was performed using the UPGMA (unweighted pair group method with arithmetic mean clustering algorithm) to calculate the dendrograms.
3
Assessing Microbial Diversity via DGGE Analysis
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HDA1 and HDA2-GC (GC clamp required for DGGE analysis [38 (link)], targeting the V2-V3 region [39 (link)]) were used to assess microbial diversity in each sample. The PCR products were separated in 8% polyacrylamide gels (37.5 : 1 acrylamide : bisacrylamide) with a range of 30–50% denaturing gradient (100% denaturant consisted of 7 M urea and 40% deionized formamide) cast with Bio-Rad's Model 475 gradient delivery system (BioRad, Hercules, CA, USA). The electrophoresis was performed in TAE 0.5X buffer for 5 h at a constant electric current of 125 mA and a temperature of 60°C with the DCode Mutation Detection System (Bio-Rad, Hercules, CA, USA). Clustering analysis was performed using the UPGMA (unweighted pair group method with arithmetic mean clustering algorithm) to calculate the dendrograms.
4
DGGE Protocol for DNA Profiling
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DGGE was performed using Dcode Mutation Detection System (Bio-Rad Laboratories Ltd., Hertfordshire, UK). PCR products were electrophoresed in 0.5 × TAE buffer (1 × TAE buffer is 0.04 M Tris base, 0.02 M sodium acetate, and 10 mM EDTA, pH 7.4) on 8% acrylamide gel containing 25–50% denaturating gradient of formamide and urea. DGGE was conducted at 60°C for 5 h at voltage of 200 V. The gel was stained with SYBR Green I Nucleic acid gel stain (Cambrex Bio Science Rockland, USA), photographed and analyzed for DGGE band profile with a UV gel documentation system (Bio-Rad Laboratories Inc., CA, USA).
5
Soil Bacterial Community Analysis by DGGE
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Total DNA was extracted from 0.5 g soil samples using a GeneMATRIX Soil DNA Purification Kit (Eurx, Poland) as described in the manufacturer's instruction. The 16S rRNA gene fragment was amplified using the primers (GC-clamp)-F338 and R518 (Muyzer et al., 1993 (link)). Detailed information about this procedure was described in a previous paper (Cycon et al., 2013 (link)). The electrophoresis of the amplification products was performed in 8 % (w/v) polyacrylamide gel (37.5:1 acrylamide:bis-acrylamide) in the presence of a linear denaturing gradient that ranged from 40 to 70 % using a DCode Mutation Detection System (Bio-Rad, USA). In turn, a G BOX F3 System (Syngene, UK) was used to visualize the patterns of the bands that were obtained. Detailed information about the DGGE procedure was described in a previous paper (Cycon et al., 2016 (link)). The band patterns that were obtained from the DGGE analysis were analyzed using BioNumerics software ver. 7.5 (Applied Math, Belgium). The unweighted pair-group method and the arithmetic averages (UPGMA) were used to construct the phylogenic dendrograms. The biodiversity of a soil bacterial community was expressed as the Shannon-Wiener index (H'), richness (Rs) and evenness (Eh), which were calculated using the equations that were described in a previous paper (Cycon et al., 2013 (link)).
6
Prostate Cancer EPS DGGE Analysis
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Denaturing gradient gel electrophoresis analysis was performed for the EPS specimens of prostate cancer and BPH, using the Bio-Rad Dcode mutation detection system (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. DNA fragments with different sequences were separated in 8% polyacrylamide (acrylamide: bisacrylamide = 37.5: 1; w/v) gels in 1× TAE buffer with 200 ng of each PCR product. A 30–60% denaturant gradient (100% denaturant was 7 mol/l urea and 40% (v/v) deionized formamide) was applied in the DGGE electrophoresis, which was performed in 1×TAE buffer at 180 V at 60°C for 4 h. Subsequently, the gels were washed with ultrapure water and stained with 5% Goldview dye for 30 min and photographed. DGGE graphs were digitized by Quantity One Analysis software (Gene Genius).
7
Soil Bacterial Diversity via DGGE
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The genetic diversity of soil bacteria was analyzed using the amplification of the 16S RNA gene fragment with the primers (GC-clamp)-F338 and R518 (Muyzer et al., 1993 (link)) with the use of a previously described method (Cycon et al., 2013 (link)). The electrophoresis was run in polyacrylamide gel (8% w/v, 37.5:1 acrylamide:bis-acrylamide) with a linear gradient of denaturant urea (40%−70) using a DCode Mutation Detection System (Bio-Rad, USA). The patterns of the DGGE bands were visualized using a G BOX F3 System (Syngene, UK) (Cycon et al., 2016 (link)).
8
DGGE Gel Electrophoresis Protocol
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DGGE was performed using Dcode Mutation Detection System (Bio-Rad Laboratories Ltd., Hertfordshire, UK). PCR products were electrophoresed with 0.5 × TAE buffer (1 × TAE buffer is 0.04 M Tris base, 0.02 M sodium acetate, and 10 mM EDTA [pH 7.4]) on 8% acrylamide gel containing 25 to 50% denaturating gradient of formamide and urea. DGGE was conducted at 60°C for 5 h at voltage of 200 V. The gel was stained with SYBR Green I Nucleic acid gel stain (Cambrex Bio Science Rockland, USA), photographed and analyzed for DGGE band profile with a UV gel documentation system (Bio-Rad Laboratories Inc., CA, USA).
9
Profiling Microbial Diversity via DGGE
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The PCR products of the V3 region for the 16S rRNA genes were electrophoretically separated by DGGE using the Bio-Rad Dcode mutation detection system (Hercules, CA, USA) according to the manufacturer instructions. Next, 40 mL of each PCR product was loaded on 10% (w/v) polyacrylamide gels with a denaturing gradient from 30-60% and containing 12.6-25.2% (w/v) urea and 12-24% (v/v) deionized formamide (Sangon Biotech). Electrophoresis was performed in 1X TAE buffer for 10 min at a constant voltage 200 V at 60°C, and then at 85 V for 16 h. The gel was stained with 0.5 mL/mL ethidium bromide for 20 min, rinsed with deionized water for 20 min, and photographed. Characteristic bands were removed from the gel. The small gel pieces were rinsed with 100 mL 70% precooled ethanol 3-4 times, and then 50 mL double-distilled H 2 O was added to the gel pieces after they were air-dried and soaked for approximately 10 h at 4°C. PCR was conducted for the recovered DNA fragments under the same conditions as were used for the V3 region, and detected and purified by DGGE. Next, the recovered DNA fragments were amplified in the V3 region-specific primers F341 (5'-ACG GGG GGC CTA CGG GAG GCA G-3') and R518 (5'-ATT ACC GCG GCT GCT GG-3') without a GC-clamp. The PCR products were sequenced by Sangon Biotech.
10
Fungal Community Analysis by DGGE
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The PCR amplicons for fungi with GC clamp were separated on an 8% polyacrylamide gel (acrylamide:bisacrylamide, 39.5:1) containing 30-60% denaturant (urea). Electrophoretic separation was performed in 1X TAE buffer (Tris, acetic acid, EDTA, pH 8.0) in a DCode Mutation Detection System (Bio-Rad). The electrophoresis was run for 17 h at 40V and 60°C. The gel was stained with SYBR Green (1:10,000; Invitrogen) for 15 min and destained in Mil-liQ water for 30 min. The gel was visualized under UV light and photographed. Densitometric analysis was performed using Quantity One 1-D software (Bio-Rad). The Shannon biodiversity index was calculated as described previously (Ziembińska et al., 2009) .
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