PCR amplification of the ARHD genes from the plasmid clones was performed in 96‐well PCR plates in a final volume of 25 μl using the primers M13F (5′ CGC CAG GGT TTT CCC AGT CAC GAC 3′) and M13R (5′ TTT CAC ACA GGA AAC AGC TAT GAC 3′). PCR reaction contained 1X Taq buffer (Invitrogen), 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP mix (GE Healthcare), 0.4 μmol/L of each primer, 2 U Platinum Taq DNA polymerase (Invitrogen) and 1 μl (10 ng/μl) of plasmid DNA. The reaction was conducted in an Eppendorf Mastercycler Gradient (Eppendorf Scientific, New York, USA) and the amplification program consisted of an initial denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 20 s and extension at 72°C for 90 s, and a final extension step at 72°C for 5 min. The PCR products were purified using the Illustra GFX 96 PCR Purification Kit (GE Healthcare Life Sciences) and checked on 1% agarose gel electrophoresis (Fisher Scientific, MA). The purified PCR products were then used as template for sequencing reaction using Big Dye kit (Life Technologies) and the primers M13R, according to manufacturer's guidelines. The sequencing of ARHD gene library was performed using the ABI 3500 XL platform (Applied Biosystems® 3500 XL).
Taq buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
Taq buffer is a solution designed for use with Taq DNA polymerase in polymerase chain reaction (PCR) applications. It provides the necessary ionic conditions and pH to enable the optimal activity of Taq polymerase during DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Taq polymerase, by Thermo Fisher Scientific (12 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (11 mentions)
Mgcl2, by Thermo Fisher Scientific (9 mentions)
Dntps, by Thermo Fisher Scientific (5 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (3 mentions)
Qiaquick pcr purification kit, by Qiagen (3 mentions)
Dntp mix, by GE Healthcare (2 mentions)
Mastercycler gradient, by Eppendorf (2 mentions)
Taq dna polymerase, by Roche (2 mentions)
C1000 touch thermal cycler, by Bio-Rad (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Silica membrane based kit, by Qiagen (2 mentions)
Dna blood mini kit, by Qiagen (2 mentions)
Illustra gfx 96 pcr purification kit, by GE Healthcare (1 mentions)
Bigdye kit, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Sybr safe dna gel staining, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Promega (1 mentions)
Facsaria 3, by BD (1 mentions)
Nanovue spectrophotometer, by GE Healthcare (1 mentions)
37 protocols using taq buffer
1
Amplification and Sequencing of ARHD Genes
2
Single-Cell Sorting and Sequencing of Antibody Genes
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Cell suspensions were prepared as for flow cytometric analysis. SWHEL GC cells, plasmablasts (Fig. 4 ), and non-GC cells (FasloCD38hi) from Pik3cdGOF⋅SWHELRag1−/−:H3Xtg chimeras were single-cell sorted into 96-well plates (Thermo Fisher Scientific) using a FACSAriaIII (BD Biosciences). Each well contained 10× Taq Buffer (Invitrogen), 10 mg/ml proteinase K (Promega), 10% Tween-20, 10 mM Na2EDTA, and distilled H2O (Baxter) in a total volume of 10 µl. proteinase K digestion was performed by heating plates to 56°C for 40 min, followed by 95°C for 8 min. PCR was then performed with Taq DNA polymerase (Invitrogen) and deoxyribonucleotide triphosphates (Sigma-Aldrich). For primary PCR, HyHEL10 primary forward and reverse sequencing primers were used. Primary PCR conditions were as follows: 94°C for 3 min, followed by 34 cycles of 95°C for 15 s, 55°C for 1 min, and 72°C for 1 min. A 1:10 dilution of the primary PCR product was then transferred into a fresh 96-well plate for secondary PCR. For secondary PCR, HyHEL10 secondary forward and reverse sequencing primers were used. Secondary PCR conditions were as follows: 94°C for 3 min, followed by 35 cycles of 95°C for 15 s, 62°C for 40 s, and 72°C for 1 min. PCR product purity was determined using SYBR Safe DNA gel staining (Thermo Fisher Scientific). Sanger sequencing was performed by Genewiz.
3
16S rRNA Gene Amplification Protocol
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Amplification analysis of the 16S rRNA gene were developed by polymerase chain reactions (PCR), using 27f and 1401r (Lane 1991 ) as primers at the following conditions: DNA 6.5 µL, Taq Buffer 5 µL; MgCl2 (2.5 nM) 1.5 µL, dNTP’s (2.5 nM) 0.4 µL, primers (20 µM) 1 µL, Taq DNA polymerase (Invitrogen) 0.4 U/µL. The PCR amplifications were done using 40 cycles of 2 min at 94 °C, 1 min at 50 °C and 3 min at 72 °C, in a Veriti Applied Biosystems ABI thermal cycler. PCR products (2 µL) were quantified at NanoVue spectrophotometer (GE Healthcare).
4
Mitochondrial DNA Extraction and Sequencing
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Total DNA was extracted from one worker per colony using a phenol-chloroform protocol [27 ]. Fragments of the mitochondrial genes cytochrome c oxidase I (COI) [28 (link)] and cytochrome b (Cytb) [29 (link)], commonly used for this type of study, were amplified. All polymerase chain reactions (PCR) contained template DNA (50 ng), 1X of Taq buffer (Invitrogen, USA), 250 μM of each dNTP, l.0 μM of each primer, 2.5 mM of MgCl2 and 1 U of Platinum Taq DNA polymerase (Invitrogen, USA) in a final volume of 25 μL. The PCR conditions for all gene regions were as follows: initial denaturation at 94°C for 5 minutes, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 50°C and extension at 72°C for 1 min, and completed with an additional extension step at 72°C for 10 min. The PCR products were electrophoresed in agarose gel stained with GelRedTM. The PCR product was purified using the Illustra ExoProStar 1-Step Kit (GE Healthcare, Buckinghamshire, UK). The mitochondrial genes were sequenced in both directions with the same amplification primers using the BigDye v 3.0 Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Carlsbad, CA, USA) in an automated sequencer ABI 3730 XL (Applied Biosystems).
5
Metagenomic ARHD Gene Amplification
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The mangrove metagenomic DNA was used as template for ARHD gene PCR‐based amplification using the degenerate primers: ARHDf (TTY RYI TGY AII TAY CAY GGI TGG G) and ARHDr (AAI TKY TCI GCI GSI RMY TTC CA) (Bellicanta, 2005 ). These primers were designed to flank a highly conserved region of the alpha subunit from ARHD gene, with an expected amplicon size ranging between 300 and 329 bp. The PCR reaction was prepared to a final volume of 50 μl containing 1X Taq buffer (Invitrogen), 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP mix (GE Healthcare), 1.2 μmol/L of each primer, 1 U Platinum Taq DNA polymerase (Invitrogen) and 2 μl (12 ng/μl) of eDNA. The reaction was conducted in an Eppendorf Mastercycler Gradient (Eppendorf Scientific, New York, USA) and the program consisted of an initial denaturation at 97°C for 3 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 1 min and extension at 72°C for 1 min, and a final extension step at 72°C for 5 min. PCR products were separated on 1% agarose gel (Fisher Scientific, MA) in 1X TAE buffer, using the molecular weight marker 1 Kb Plus DNA Ladder (Invitrogen) for size estimation and observed under UV light using a ImageQuant LAS 4000 system (GE Healthcare Life Sciences).
6
Amplification of Bacterial blaZ Gene
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Whole genomic DNA was isolated as described by Pospiech & Neumann (1995) . PCR amplification of the internal region of the blaZ gene was carried out using primers designed by Vesterholm-Nielsen et al. (1999) with the following sequences: blaZ primer1: AAG AGA TTT GCC TAT GCT TC and blaZ primer2: GCT TGA CCA CTT TTA TCA GC. The PCR reaction mixture (25 μl) contained 1-μM of primer 1 and 2, 0•2-mKm of dNTP, 0•25 μl of Taq buffer 10x, 0•25 U of Taq polymerase (Invitrogen CA, USA) and 25 ng of DNA template. Amplification was carried out on thermal cycler Techne TC 3000 G (Techne Inc. NJ, USA) using a program as follows: an initial 5-min denaturation step at 94 °C, followed by 35 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 55 °C, and 1 min of extension at 72 °C; with a final extension step at 72 °C for 10 min (Haveri et al. 2005) . PCR products were analysed by electrophoresis on ethidium bromide-stained 2% agarose gels (Biodynamics, Buenos Aires, Argentina). A positive (Staph. aureus ATCC 29213) and a negative control (without DNA template) were included in each PCR run.
7
Nested PCR for Adenovirus Hexon Protein
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Nested PCR was used to amplify the hexon protein-coding region of adenovirus. The 50 μL reaction mix contained 5 μL extracted DNA, 1 × Taq Buffer (Applied Biosystems), 2.5 mM MgCl2 (Applied Biosystems), 0.2 mM dNTP (Sigma-Aldrich), 0.5 μM of forward and reverse primers (AdvF 1 st and AdvR 1st; Table S1), and 1U Taq DNA polymerase (Roche Diagnostics). The PCR was initiated at 94 °C for 3 min, followed by 40 cycles at 94 °C for 20 s, 59 °C for 30 s, and 72 °C for 1 min, and a 5 min final extension at 72 °C. Five microliters of the PCR product from the first-round amplification were used as the template for nested PCR as described above but with different forward and reverse primers (AdvF 2nd, AdvR 2nd; Table S1). The fragments were purified using a QIAquick PCR purification kit (Qiagen) according to the manufacturer’s protocol, and the purified amplicons were sent for sequencing to GATC Biotech, Constance, Germany.
8
Nested PCR Amplification of DNA and cDNA
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Extracted DNA and cDNA samples were amplified by nested PCR in triplicate. Briefly, touch-up gradient PCR using primers SISP3 and SIS3 (Table S1) was used as the first-round amplification in a 50 μL reaction mix containing 6.5 μL template, 1 × Taq buffer (Applied Biosystems), 2 mM MgCl2 (Applied Biosystems), 0.5 mM dNTP (Sigma-Aldrich), 1 U Taq DNA polymerase (Roche Diagnostics), and 0.8 μM of each primer. The PCR reaction was performed for one cycle at 94 °C for 3 min, followed by 12 cycles touch-up PCR with 94 °C for 30 s and 20 °C for 190 s (2 °C increase per cycle), followed by 30 cycles of 94 °C for 30 s, 48 °C for 30 s, and 68 °C for 2 min, and with a 5 min final extension at 68 °C. Five microliters of the first-round PCR product were further amplified with the primers SISP2 and SIS2 (Table S1) in a 50 μL reaction mix containing 1 × Taq buffer (Applied Biosystems), 2 mM MgCl2 (Applied Biosystems), 0.5 mM dNTP (Sigma-Aldrich), 1 U Taq DNA polymerase (Roche Diagnostics), and 0.8 μM of each primer. The PCR was performed with an initial denaturation at 94 °C for 3 min followed by 45 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 2 min, and with a 5 min final extension at 68 °C. The triplicates were pooled, and the PCR products were visualized by 1.5% agarose gel electrophoresis.
9
PCR Assay for Detecting L. monocytogenes
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A formerly developed PCR procedure [29 (link),30 (link)] targeting prfA of L. monocytogenes was used as a standard for comparison with the novel LAMP assay. The master mix comprised 3 mM of MgCl2, 150 mM dNTPs, 0.25 μM of each primer (LIP1 and LIP2, Table S1 ), 1 U Taq DNA Polymerase, 1× Taq Buffer (Thermo Fisher Scientific) and 0.8 ng of DNA. The amplification was performed in a T100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) and PCR products were resolved on 0.8% (w/v) gel electrophoresis.
10
DNA Amplification and Visualization
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Taq DNA polymerase, Taq buffer and MgCl2, dNTPs, and digestion enzymes were purchased from Thermo Scientific, (EU) Lithuania. Primers, ladders, and agarose were obtained from Invitrogen by Life Technologies, CA, USA. The GelredTM staining was supplied by Biotium, Inc., Hayward, CA, USA. Proteinase K was obtained from Roche Diagnostic, Mannheim, Germany.
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