Five isolates of mycoparastic fungi were isolated from the gathered sclerotia of S. sclerotiorum. The morphological characteristics of all five isolates were identified based on their dark-yellowish pigmentation on the Petri dish, the presence of orange-coloured sporodochia, conidiophores, conidial colour, and size [11 (link),12 ]. The 9 mm mycelial plugs of isolated mycoparasitic fungi were positioned in 250 mL Erlenmeyer flasks containing 150 mL potato dextrose broth and incubated at 23 °C for 20 days. Later, the mycelial mats of all the isolates were collected, dried on sterilized paper, and finely crushed using liquid nitrogen with a mortar and pestle. Genomic DNA was obtained from the pure culture of mycoparasitic fungi using cetyl trimethyl ammonium bromide (CTAB) [57 (link)]. PCR amplification was carried using ITS 1 (sequence: 5′- TCCGATGGTGAACCTGCGG-3′) and ITS 4 primers (sequence: 5′TCCTCCGCTTATTGATATGC-3′) [58 ]. The master cycler gradient PCR (Eppendorf) was executed with a 25 µL reaction volume with an initial template DNA step of 95 °C for 10 min, denaturation at 94 °C for 30 s, annealing step at 60 °C for 1 min, extension step at 72 °C for 1 min and the final extension step at 72 °C for 10 min, followed by 35 cycles and held at and 4 °C.
Mastercycler gradient pcr
Manufactured by Eppendorf
Sourced in Germany
The Mastercycler Gradient PCR is a thermal cycler designed for polymerase chain reaction (PCR) processes. It features a gradient function that allows for the optimization of annealing temperatures across multiple samples simultaneously. The Mastercycler Gradient PCR provides precise temperature control and consistent results for a wide range of PCR applications.
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Lab products found in correlation
Maxima sybr green qpcr master mix, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Qiaamp mini kit, by Qiagen (2 mentions)
Master mix go taq g2, by Promega (2 mentions)
Ribopure blood kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Qiaamp dna blood kit, by Qiagen (1 mentions)
Taq dna polymerase, by Takara Bio (1 mentions)
Maxima reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by Merck Group (1 mentions)
Gel documentation system, by Bio-Rad (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Verso cdna kit, by Thermo Fisher Scientific (1 mentions)
12 protocols using mastercycler gradient pcr
1
Isolation and Characterization of Mycoparasitic Fungi
2
Microbial Community Profiling using PCR
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TransGen AP221-02: TransStart Fastpfu DNA Polymerase was used for PCR. The primer sequences were as follows: 338F: ACTCCTACGGGGAGCA, 806R: GGACTACHVGGGTWTCTAAT. The PCR system consisted of the following reaction volume: 25 μl, 12.5 μl 2 × Taq-PCR-MasterMix, 3 μl BSA (2 ng/μL), 1 μl (5 μmol/l) of each primer, 2 μl template DNA, and 5.5 μl ddH2O, and was amplified on the Mastercycler Gradient PCR (Eppendorf, Germany). The circulation parameter was 95°C for 5 min, then it was cycled at 95°C for 45 s, 55°C for 50 s, 72°C for 45 s, and finally it was prolonged at 72°C for 10 min. Each sample was repeated 3 times. The AxyPrepDNA gel recovery kit was used to cut and recover the PCR products, and Tris–HCl elution was performed. The PCR products of the same sample were mixed and detected by 2% agarose gel electrophoresis.
3
Total RNA Isolation and Purification from Whole Blood
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Total RNA from whole blood was isolated and purified using RibopureTM- blood Kit (Ambion inc. Texas, USA) following the manufacturer’s protocol. RNA integrity was verified by agarose gel electrophoresis/ ethidium bromide staining and O.D. 260/280 absorbance ratio >1.95. RNA was treated with DNase I (Ambion inc. Texas, USA) before cDNA synthesis to avoid DNA contamination. One microgram of total RNA was used to prepare cDNA. cDNA synthesis was performed using the Verso cDNA Synthesis Kit (Thermo scientific, Lithuania, EU) according to the manufacturer’s instructions using Mastercycler Gradient PCR (Eppendorf, Germany).
4
Genotyping CTLA4 and TG Genes
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Genomic DNA was extracted from whole blood using ‘QIAamp DNA Blood Kit’ (QIAGEN Inc., Valencia, CA 91355, USA) according to the manufacturer’s instructions. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used to genotype exon 1 +49A/G and 3’ UTR CT60A/G polymorphisms of CTLA4 gene and E33 polymorphism of TG gene and amplification was performed using Mastercycler Gradient PCR (Eppendorf, Germany). according to the protocol: 95°C for 10 minutes followed by 30 cycles of 95°C for 30 seconds, primer (S2 Table ) dependent annealing for 30 seconds, and 72°C for 30 seconds. The amplified products were checked by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.
Restriction enzymes (New England Biolabs, Beverly, MA) used for digesting amplicons of exon 1 +49A/G and 3’ UTR CT60A/G of CTLA4 gene and E33 polymorphism of TG gene are given inS2 Table . 15 μL of the amplified products were digested for 16 hours at 37°C with 5 U of the corresponding restriction enzyme. The digestion products with 100/50 base pair DNA ladder (Bioron, Ludwigshafen am Rhein, Germany) were resolved in 3.5% agarose gels stained with ethidium bromide and visualized under UV transilluminator.
Restriction enzymes (New England Biolabs, Beverly, MA) used for digesting amplicons of exon 1 +49A/G and 3’ UTR CT60A/G of CTLA4 gene and E33 polymorphism of TG gene are given in
5
ISSR Molecular Marker Screening Protocol
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ISSR primers used in this study were synthesized by Beijing Dingguo Changsheng Biotechnology Co., Ltd (China), according to the primer set published by the University of British Columbia (UBC). One hundred ISSR primers were initially screened, and twelve that yielded bright and discernible bands, were used for the analysis of all 126 samples (Table 1 ). Fifteen or sixteen individuals from each population were used for the primer screening, and PCR amplifications were repeated for working primers to check the stability and reproducibility of ISSR fragments. PCR was performed in 25 μl reactions containing 1.75 mM MgCl2, 0.25 mM dNTPs, 1 U Taq DNA polymerase (TaKaRa), 0.2 μM primers and 10 ng DNA templates. PCR amplifications were performed in the Mastercycler Gradient PCR (Eppendorf, Germany) with the following program: initial denaturation at 94°C for 5 min; 40 cycles of 94°C for 50 s, appropriate annealing temperature (see Table 2 ) for 45 s, 72°C for 1 min; and final synthesis at 72°C for 10 min. A negative control with no DNA added was included in each PCR run. Amplification products were separated with 1.5% agarose gels (1×TAE buffer) at 80 V for 1.5 h, stained with ethidium bromide (0.5 μg/ml), and photographed under UV light using an EC3 Gel Documentation System (UVP, USA).
6
Gene Expression Analysis by RT-PCR
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Total RNA was extracted and reverse-transcribed using Maxima Reverse transcriptase (Thermo Scientific). RT-PCR or qRT-PCR was done to assess gene expression with gene-specific qRT-PCR primers (Supplementary Table 4
7
Genetic Polymorphism Genotyping
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Genotyping of DNA repair genes (XRCC1, XRCC2, XPD, APE1, and hOGG1), carcinogen detoxifying genes (GSTM1, GSTP1, GSTT1, and NAT), and oxidative stress-related genes (SOD1, SOD2, and catalase) were performed by the polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method with appropriate primer sets. The primers were designed to amplify the regions of DNA that contain polymorphic sites of interest. PCR amplification was carried out separately under different conditions in 20 μL reaction mixtures containing 1X PCR buffer 0.2 mM each dNTP, 10 picomoles of each of selected primers, 1U Taq DNA polymerase (GeNei, Merck Bioscience), and 100 nanogram of purified genomic DNA template. The obtained mixtures were imperilled to PCR amplification with a Master cycler Gradient PCR (Eppendorf). The PCR products, obtained for each reaction were analyzed using 2% agarose gel electrophoresis (1X TAE buffer), which were stained with ethidium bromide (10 mg/mL) and later envisioned under UV Transilluminator and saved (photographed) in gel documentation system (Bio Rad Laboratories). After confirmation of DNA amplification, each PCR product was digested with an appropriate restriction enzyme for genotyping.
8
Genomic DNA Extraction and COI Gene Amplification
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Total genomic DNA was isolated from the crabs' muscles using QIAamp mini kit (Qiagen, GmbH, Germany) and the manufacturer's protocol was followed. The partial coding region of the COI gene was amplified by PCR. The PCR reaction mixture consisted of 12.5 μl (1X) colorless Master Mix Go Taq® G2 (Promega Corporation-Madison, WI, USA), 1 μl (10 pmol/ μl) of each primer pair and 2 μl (10 ng/ μl) of DNA template then complete to 25 μl using nuclease-free double distilled water. The primer pair used were forward primer CrustF1 (5'-TTTTCTACAAATCATAAAGACATTGG-3') and reverse primer HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') as mentioned by Costa et al. (2007) . Primers were synthetized by Metabion international AG, Germany. The amplification reaction was performed using Mastercycler gradient PCR (eppendorf, Germany). The thermal cycle conditions of PCR was beginning with initial denaturation of 60s at 94 °C then five cycles of 30s at 94 °C, 90s at 45 °C, and 60s at 72 °C; 35 cycles of 30s at 94 °C, 90s at 51 °C, and 60s at 72 °C; followed by a final extension of 5 min at 72 °C. The amplified products were separated according to base pair (bp) size using 2 % agarose gel electrophoresis stained by ethidium bromide. The gel was then visualized, imaged and analyzed using the Gel Documentation system with UV light box and GeneSys software (Syngene, Synoptics Ltd, England).
9
Confirming ureA Gene Presence in Plasmids
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The presence of ureA gene in extracted plasmids was confirmed by conventional polymerase chain reaction (PCR) technique. The specific oligonucleotide primers for H. pylori ureA gene were designed using Gene Runner software version 3.05 and the primer sequences was aligned to a query sequence of GenBank data using BLAST (Basic Local Alignment Search Tool) (Table 1). 25 μL of the reaction mixture containing 20 to 50 ng of purified plasmid DNA, 2.5 μL of 10x Taq buffer (100 mM Tris-HCl, pH 8), 100 μM dNTPs, 1.2 μM forward and reverse primers, 1.25 mM MgCl 2 , and 2.5 U Taq DNA polymerase (all Invitro-gen, USA) was added to the 0.2 mL micro-tubes. The content was mixed, and the proliferation was performed in a Mastercycler Gradient PCR (Eppendorf, Germany) following the program given below: initial denaturation (94°C, 5 min), followed by 35 cycles, including denaturation (94°C, 1 min), annealing (66°C, 1 min), extension (72°C, 1 min), then the final extension step at 72°C for 10 min and hold at 4°C. PCR products were visualized by ethidium bromide staining on 2% agarose gel electrophoresis in TBE 1X buffer according to the procedure as mentioned above.
10
DNA Extraction and COI Amplification
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DNA was extracted from specimens" muscle using QIAamp mini kit (Qiagen, GmbH, Germany) following the manufacturer"s protocol. Eventually, three samples of COI were amplified by mixing 12.5 μl (1X) colorless Master Mix Go Taq® G2 (Promega Corporation-Madison, WI, USA), 1 μl (10 pmol/ μl) of forward primer CrustF1 (5'-TTTTCTACAAATCATAAAGACATTGG-3') and 1 μl (10 pmol/ μl) of reverse primer HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') described by Costa et al. (2007) , and 2 μl (10 ng/ μl) of DNA template. The reaction volume was adjusted to 25 μl using nuclease-free double distilled water. Primers were synthesized by Metabion international AG, Germany. The amplification reaction was carried out using Mastercycler gradient PCR (eppendorf, Germany). The thermal cycle conditions consisted of initial denaturation of 60 s at 94 °C; five cycles of 30 s at 94 °C, 90 s at 45 °C, and 60 s at 72 °C; 35 cycles of 30 s at 94 °C, 90 s at 51 °C, and 60 s at 72 °C; followed by a final extension of 5 min at 72 °C. PCR product was detected and visualized using 2 % agarose gel electrophoresis stained by 25 μg of ethidium bromide. The gel was then imaged and analyzed (template size detection) using Gel Documentation system with UV light box and GeneSys software (Syngene, Synoptics Ltd, England).
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