Individual worms were placed in PCR tubes containing 4 μL 1× Thermopol® reaction buffer (NEB) and 0.5 mg/mL proteinase K buffer (NEB) and incubated at −80 °C for 30 min, then incubated at 65 °C for 1 h and 95 °C for 15 min to inactivate proteinase K. SW-PCR was initiated by adding 20 μL of PCR mix that contained primer pairs, dNTPs, and 1× Thermopol® reaction buffer for a reaction volume of 24 μL. The PCR cycle consisted of the following steps: 55 °C for 60 s, 94 °C for 5 min 15 s, 55 °C for 15 s, 72 °C for 1 min (35 cycles), and 72 °C for 5 min (total reaction time 1 h and 30 min).
Thermopol reaction buffer
Manufactured by New England Biolabs
Sourced in United States, China
ThermoPol Reaction Buffer is a specialized buffer solution developed by New England Biolabs for use in various molecular biology applications. It is designed to provide optimal conditions for the activity of thermostable DNA polymerases, enabling efficient DNA amplification and synthesis at elevated temperatures.
Automatically generated - may contain errors
Lab products found in correlation
Bst dna polymerase large fragment, by New England Biolabs (10 mentions)
Mgso4, by New England Biolabs (9 mentions)
Betaine, by Merck Group (7 mentions)
Bst dna polymerase, by New England Biolabs (7 mentions)
Taq polymerase, by New England Biolabs (6 mentions)
Dntps, by Takara Bio (5 mentions)
Taq dna polymerase, by New England Biolabs (5 mentions)
Dntps, by New England Biolabs (4 mentions)
Deoxyribonucleotide triphosphate, by New England Biolabs (4 mentions)
Evagreen, by Biotium (4 mentions)
Bst 2.0 dna polymerase, by New England Biolabs (3 mentions)
Mgso4, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by New England Biolabs (3 mentions)
Nebuffer 2, by New England Biolabs (3 mentions)
Nebuffer 3, by New England Biolabs (2 mentions)
Bst 2.0 warmstart dna polymerase, by New England Biolabs (2 mentions)
Sybr green 1, by Thermo Fisher Scientific (2 mentions)
Dnase rnase free deionized water, by Tiangen Biotech (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
60 protocols using thermopol reaction buffer
1
Single Worm DNA Extraction and Amplification
2
Maintenance of Avian Leukosis Viruses
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ALV-J (HN06), ALV-A (GD-13), ALV-B (CD08), ALV-K (GDFX0601), ALV-E (HN1301) and Salmonella enteritis (SE) were maintained in our laboratory. Escherichia coli ATCC15922 and Pseudomonas aeruginosa ATCC27853 were obtained from HuanKai Microbial (Guangzhou, China). Mycoplasma gallisepticum (MG) S6 was donated by Professor Ding (College of Veterinary Medicine, South China Agricultural University). Bst DNA polymerase, 10 × ThermoPol Reaction Buffer, dNTPs and MgSO4 were products of New England Biolabs (Beverley, MA, USA). Betaine was acquired from Sigma (St. Louis, MO, USA). Disposable Nucleic Acid Detection Strip were purchased from Ustar (Hangzhou, China). The DNA Extraction Kit, Gel Extraction Kit, and Plasmid Mini Kit were produced from OMEGA (Norcross, GA, USA).
3
Optimized LAMP System for Detecting Sugarcane Smut
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The LAMP reaction was carried out in a 25 μL mixture containing 3.75 mmol/L MgSO4 (50 mmol/L), 1.4 mmol/L dNTPs (10 mmol/L), 2.5 μL 10×ThermoPol Reaction Buffer [including 20 mmol/L Tris-HCl pH 8.8, 10 mmol/L KCl, 2 mmol/L MgSO4, 10 mmol/L (NH4)2SO4 and 0.1% Triton X-100, NEB company], 0.8 μmol/L each of FIP and BIP primers, 0.2 μmol/L each of F3 and B3 primers, 8.0 U Bst DNA polymerase (8 000 U/mL, NEB company), 1.0 μL template DNA, and ddH2O, which was complemented to a volume of 25 μL. The blank control (ddH2O), negative control (FN40 tissue culture seedlings free of fungal pathogens) and positive control (pMD19-T-Pep1 plasmid) were set for the experiment. Key compositions of the LAMP system were optimized using a single factor experiment and an orthogonal experiment to obtain an optimal LAMP system which is stable and sensitive enough for the detection of S. scitamineum.
4
Multiplex PCR for Tick-Borne Pathogens
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Standard or nested polymerase chain reaction (PCR) was employed to screen all blood samples using species- or genus-specific primers (Additional file 1 : Table S2), including A. ovis major surface protein 4 (msp4) [23 (link)], A. bovis 16S ribosomal RNA (16S rRNA) [24 (link)], A. capra citrate synthase (gltA) [25 (link)], A. phagocytophilum 16S rRNA [26 (link)], A. marginale msp4 [23 (link)], Babesia ovis 18S ribosomal RNA (18S rRNA) [27 (link)], B. bovis spherical body protein 4 (SBP4) [28 (link)], B. bigemina rhoptry-associated protein 1a (rap1a) [28 (link)], B. motasi-like Lintan/Ningxian/Tianzhu rhoptry-associated protein 1b (rap1b) [17 (link)], and Theileria spp. 18S rRNA [29 (link)]. The PCR mixture consisted of 2 µl of DNA template, 0.5 µl each of forward and reverse primer (100 μM), 0.1 µl of Taq polymerase (0.5 U; New England BioLabs, USA), 0.2 µl of deoxyribonucleotide triphosphate (200 μM; New England BioLabs, USA), 1 µl of 10× ThermoPol Reaction Buffer (New England BioLabs), and double-distilled water for a total volume of 10 µl. DNA samples from the blood of animals infected with the respective pathogens, which had been collected and stored properly in previous studies, were used as positive controls. Double-distilled water was used as a negative control.
5
Isothermal DNA Amplification with Ligation
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The DNA oligonucleotides were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and purified by high-performance liquid chromatography. Taq DNA ligase (40 U μL−1), 10 × Taq DNA ligase reaction buffer (200 mM Tris-HCl, 250 mM KAc, 100 mM Mg(Ac)2, 100 mM DTT, 10 mM NAD, 1% Triton X-100, pH 7.6), Bst 2.0 DNA polymerase (8 U μL−1) and 10 × ThermoPol reaction buffer (200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Tween-20, pH 8.8) were all purchased from New England Biolabs (Beijing, China). SYBR Green I was obtained from Generay Biotech. Co., Ltd. (Shanghai, China). DNA marker, 6 × loading buffer and dNTPs were purchased from Takara (Dalian, China). TIANamp Genomic DNA Kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The nuclease-free water was purchased from Thermo Fisher Scientific Inc (Vilnius, Lithuania) and used in all ligation reaction and ligation-initiated LAMP assays. All the reagents were of analytical grade and used without further purification. All solutions were prepared with ultrapure water from Millipore Milli-Q water purification system (Millipore, USA). The detailed oligonucleotide sequences were listed in Table S1 (Supporting Information).
6
Bacterial 16S rRNA Gene Amplification and DGGE
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The V3 regions of bacterial 16S rRNA genes were PCR amplified using primers 341f-GC and 518r (67 (link)). Each reaction mixture contained 19.75 µl of UV-treated water, 2.5 µl of 10× ThermoPol reaction buffer (New England BioLabs), 0.05 µl of deoxynucleoside triphosphates (dNTPs) (100 mM), 0.05 µl of forward primer 341f-GC (100 µM), 0.05 µl of reverse primer 518r (100 µM), 1.5 µl of bovine serum albumin (BSA) (10 mg/ml), 0.25 µl of Taq DNA polymerase (5 U/µl) (New England BioLabs), and 1 µl of DNA template purified from each gradient fraction. The PCR conditions were initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, followed by a final extension at 72°C for 7 min. All PCR products were analyzed on 1% agarose gels prior to DGGE.
Five microliters of each PCR product was loaded onto a 10% polyacrylamide gel with a denaturing gradient of 30 to 70%. Gels were run at 60° C for 14 h at 85 V (DGGEK-2001-110; C.B.S. Scientific, San Diego, CA) as described previously (43 ). A custom DGGE ladder was loaded into the two outside wells of the gel for subsequent normalization. Gels were stained for 45 min with SYBR green I nucleic acid gel stain (Thermo Fisher) and rinsed once in water prior to imaging. Gel images were taken with a Pharos Plus molecular imager system (Bio-Rad).
Five microliters of each PCR product was loaded onto a 10% polyacrylamide gel with a denaturing gradient of 30 to 70%. Gels were run at 60° C for 14 h at 85 V (DGGEK-2001-110; C.B.S. Scientific, San Diego, CA) as described previously (43 ). A custom DGGE ladder was loaded into the two outside wells of the gel for subsequent normalization. Gels were stained for 45 min with SYBR green I nucleic acid gel stain (Thermo Fisher) and rinsed once in water prior to imaging. Gel images were taken with a Pharos Plus molecular imager system (Bio-Rad).
7
5-hmU Chemical Mapping Protocol
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Chemical mapping of 5-hmU as carried out following the previously described by Kawasaki et al. chemical conversion method [54 (link)] with some modifications.
Briefly, sheared genomic DNA was used as input, and end prep and adapter ligation were carried out using the NEBNext Ultra II DNA Library Prep Kit. After the ligation step, DNA was purified using AMPure XP beads and eluted in 50 µL of H2O. DNA denaturation was performed by adding NaOH to a final concentration of 0.05 M and incubating at 37 °C for 30 min. Oxidation was carried out by adding 2 µL of KRuO4 solution (15 mM in 0.05 M NaOH) for the “harsh oxidation” condition and 2 µL of KRuO4 solution (1.5 mM in 0.05 M NaOH) for the “mild oxidation condition”, then incubating for 30 min at room temperature. Oxidized DNA was purified using AMPure XP beads and extension was carried out by mixing 13.5 µL DNA, 1.6 µL 100 mM MgSO4, 2 µL NEB Index Primer, 2 µL 10 × ThermoPol Reaction Buffer (NEB), 0.5 µL 10 mM dNTP mix, and 0.4 µL Bst DNA Polymerase, Large Fragment (NEB), then incubating for 1 h at 37 °C. PCR amplification was carried out using the NEB Ultra DNA Library Prep Kit, with 12 cycles of PCR. Final libraries were purified using AMPure XP beads.
Briefly, sheared genomic DNA was used as input, and end prep and adapter ligation were carried out using the NEBNext Ultra II DNA Library Prep Kit. After the ligation step, DNA was purified using AMPure XP beads and eluted in 50 µL of H2O. DNA denaturation was performed by adding NaOH to a final concentration of 0.05 M and incubating at 37 °C for 30 min. Oxidation was carried out by adding 2 µL of KRuO4 solution (15 mM in 0.05 M NaOH) for the “harsh oxidation” condition and 2 µL of KRuO4 solution (1.5 mM in 0.05 M NaOH) for the “mild oxidation condition”, then incubating for 30 min at room temperature. Oxidized DNA was purified using AMPure XP beads and extension was carried out by mixing 13.5 µL DNA, 1.6 µL 100 mM MgSO4, 2 µL NEB Index Primer, 2 µL 10 × ThermoPol Reaction Buffer (NEB), 0.5 µL 10 mM dNTP mix, and 0.4 µL Bst DNA Polymerase, Large Fragment (NEB), then incubating for 1 h at 37 °C. PCR amplification was carried out using the NEB Ultra DNA Library Prep Kit, with 12 cycles of PCR. Final libraries were purified using AMPure XP beads.
8
Isothermal Pathogen Detection Assay
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The PSR assay was carried out in 25-μl reaction mixtures containing the following components: 1.0 μl Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA, USA), 2.5 μl 10× ThermoPol reaction buffer (New England Biolabs), including 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Tween 20), 0.8 M betaine (Sigma–Aldrich, Saint Louis, MO, USA), 6 mM MgSO4, 1.4 mM of each dNTP, and an appropriate amount of DNA template. The amount of primers needed for one reaction was 1.6 μM for Ft and Bt and 0.8 μM for IF and IB. At last, the reaction mixture was overlaid with a sealing agent (Patent: ZL201210371448.5 in China) to prevent cross contamination of samples by aerosol and the reactions were performed for 60 min at 65°C.
The PSR products were detected using two methods: turbidity monitoring with a real-time turbidimeter at 650 nm or direct visual detection with the aid of hydroxynaphthol blue (HNB), which is a metal ion indicator (Goto et al., 2009 (link)). For visual detection, 1 μl of HNB (Sigma–Aldrich) solution (0.2% mass fraction) was added to the reaction tube. A positive reaction is indicated by a color change from violet to sky blue, while a negative reaction remains violet. Each experiment was performed at least three times to ensure reproducibility.
The PSR products were detected using two methods: turbidity monitoring with a real-time turbidimeter at 650 nm or direct visual detection with the aid of hydroxynaphthol blue (HNB), which is a metal ion indicator (Goto et al., 2009 (link)). For visual detection, 1 μl of HNB (Sigma–Aldrich) solution (0.2% mass fraction) was added to the reaction tube. A positive reaction is indicated by a color change from violet to sky blue, while a negative reaction remains violet. Each experiment was performed at least three times to ensure reproducibility.
9
Preparation of RNase-Free EXPAR Solutions
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All DNA oligonucleotides (HPLC-grade) were synthesized by Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). HPLC-purity miRNAs, dNTPs mixture and DNA ladder marker were purchased from TaKaRa Biotechnology Co. Ltd. (Dalian, China). The sequences of all DNA oligonucleotides and miRNAs used in this study are given in Table 1 . The nickase Nt.BstNBI and Vent (exo-) DNA polymerase, 10× NEBuffer 3.1 and 10× ThermoPol Reaction Buffer were purchased from New England Biolabs (NEB, Beverly, MA, USA). SYBR Green I was purchased from Invitrogen Biotechnology Co. Ltd. (Waltham, MA, USA). Diethylpyrocarbonate (DEPC)-treated water, and Recombinant RNase Inhibitor were purchased from TaKaRa Biotechnology Co. Ltd. All other chemicals (at least analytical grade) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All DNA and miRNAs oligonucleotides sequences were diluted in 1× TE buffer (pH 8.0) to give stock solutions of 100 and 20 µM, respectively. To create and maintain an RNase-free environment, the tips and tubes are RNase-free and require pretreatment to inactivate RNase. All EXPAR solutions were prepared in DEPC-treated deionized water.
10
OTA-Specific Aptamer-Based Assay Protocol
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All the oligonucleotides were designed by mfold (www.unafold.org , accessed on 1 August 2022), synthesized and purified using HPLC by Sangon Biotech Co., Ltd., (Shanghai, China). The OTA-specific aptamer sequence was designed based on (5′-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGACA-3′), with a dissociation constant (Kd) of 0.2 μmol/L following Cruz et al. [36 (link)]. The dissolution of oligonucleotide sequences was carried out in TE buffer (10 mM Tris, 1 mM EDTA, pH = 8.0), and then the dissolved sequences were stored at 4 °C until use (Table 1 ). Deoxynucleoside triphosphate (dNTP) mixture, Bst DNA polymerase large fragment, Nb.BsrDI, 10×Cutsmart buffer (200 mM Tris-HAc, 500 mM KAc, 100 mM MgAc2, 1 g mL−1 BSA, pH = 7.9) and 10× ThermoPol reaction buffer (200 mM Tris-HCl, 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100, pH = 8.8) were supplied by New England Biolabs Inc. (Beverly, MA, USA). Ochratoxin A (OTA), ochratoxin B (OTB), aflatoxin B1 (AFB1), deoxynivalenol (DON) and zearalenone (ZEA) were obtained from Pribolab (Singapore). All chemicals were of analytical grade. Ultrapure water (>18.25 MΩ) was used to prepare all solutions. The recording of the fluorescence spectra was conducted using a Step OnePlus real-time PCR machine (Applied Biosystems, New York, NY, USA). The measurement of circular dichroism (CD) spectra was carried out with a Jasco J-1500 CD spectrometer.
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