First, reaction buffer (2.5 μL, 10× Thermopol, new England biolabs (NEB), MgSO4, 0.5 μL, 100 mM, NEB, Ipswich, MA, USA), dNTPs (1.5 μL, an equimolar mixture of dATP, dCTP, dGTP, and dTTP each at 2.5 μmoL, TaKaRa Bio (Dalian) Co., Ltd., Dalian, China), betaine (2.0 μL 10 M, Sigma, Ronkonkoma, NY, USA), primer (2.5 μL each of FIP, BIP, FLP, and BLP, 0.5 μL each of FOP and BOP, 10 μM, Shanghai Bio technology Co., Ltd., Shanghai, China), SYBR Green I (10,000×, 1:200, 0.3 μL, Coolaber Science and Technology Co., Ltd., Beijing, China), DNA template (1 μL, 10 ng/μL < initial concentration < 100 ng/μL), BST DNA polymerase large fragment (1.0 μL, 8 U/μL, NEB, Ipswich, MA, USA) were mixed with 4.7 μL of water (sterile distilled, TaKaRa Bio (Dalian) Co., Ltd., Dalian, China) in a PCR tube (200 μL, Axygen Scientific Inc., Union City, CA, USA) up to a total volume to 25 μL. The reaction system was mixed evenly and briefly centrifuged. Then the liquid surface of the amplification system was covered with 20 μL of mineral oil to prevent the volatilization of amplification products from polluting the amplification area. Last, the reaction PCR tube was placed in an amplifier (Applied Biosystems Quantum Studio 3 System, Waltham, MA, USA). A constant temperature was set at 62 °C for 40 min, generating a real-time amplification curve.
Thermopol
Manufactured by New England Biolabs
Sourced in United States
Thermopol is a thermostable DNA polymerase enzyme used in PCR (Polymerase Chain Reaction) and other DNA amplification techniques. It is isolated from the thermophilic bacterium Thermus thermophilus and exhibits optimal activity at elevated temperatures.
Automatically generated - may contain errors
Lab products found in correlation
Dntps, by Takara Bio (1 mentions)
Bst dna polymerase large fragment, by New England Biolabs (1 mentions)
Mgso4, by New England Biolabs (1 mentions)
Betaine, by Merck Group (1 mentions)
Lambda phage, by New England Biolabs (1 mentions)
Nebuffer 3, by New England Biolabs (1 mentions)
Vent exo polymerase, by New England Biolabs (1 mentions)
Cas9d10a, by New England Biolabs (1 mentions)
Na12878, by Coriell Institute for Medical Research (1 mentions)
Sqk lsk109 kit, by Oxford Nanopore (1 mentions)
Geneamp 9700, by Thermo Fisher Scientific (1 mentions)
High pure pcr product purification kit, by Roche (1 mentions)
Random primers 9, by New England Biolabs (1 mentions)
M mulv reverse transcriptase, by New England Biolabs (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Proteinase k, by Qiagen (1 mentions)
7 protocols using thermopol
1
Real-Time LAMP Amplification Procedure
2
Phage DNA Nicking and Extension
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First, 400 ng of Cas9nickase—Cas9H840A (IDT) or Cas9D10A (NEB) was pre-incubated in 1 × NEBuffer 3.1 (NEB) with 25 pmol sgRNA at 37 °C for 15 min to form Cas9-Ribonucleoprotein complex. Then, 1000 ng of Lambda Phage (NEB) was added to the tube, and a nicking reaction was carried out at 37 °C for 2 h. Nicked DNA was then extended with 3 U of Vent (exo-) Polymerase (NEB), 100–300 µM dNTPs, and 1 × Thermopol (NEB) at 72 °C for 60 min. After extension, the reaction was purified twice with AMPURE XP beads and was assessed on 1% agarose gel before proceeding with sequencing library preparation.
3
Nanopore Sequencing Library Preparation
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About 0.5–1 µg of Lambda or 3–5 µg of purified NA12878 (Coriell Institute) fragments were used as input for sequencing library preparation. A dA-tailing reaction was performed in the presence of 1 mM dATPs and 1X Thermopol (NEB) with 5U of Taq DNA Polymerase 72 °C for 5 m. The manufacturer-recommended amounts of sequencing adapters and ligation buffer from the SQK-LSK109 kit (Oxford Nanopore) were added to this reaction along with NEBNext Quick T4DNA Ligase. The ligation reaction was carried on at room temperature for 15 min before AMPURE XP beads were cleaned up. The library was quantified with a Qubit fluorometer before mixing with sequencing buffer loading beads and loading on a primed flow cell. Priming was carried out according to the manufacturer’s recommendations. Nanopore Sequencing was carried out on a MinION sequencer using FLO-FLG001 flongles or Minion R9.4 flow cells from ONT. The experiment was set up and run with live base calling performed using MinKNOW software with default configurations.
4
Molecular Cloning of M. laticollaris Peptide
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Based on the information obtained from direct peptide sequencing of Mlat1, a specific oligonucleotide was designed and used for the PCR reaction using as a template the cDNA material from a cDNA library created with M. laticollaris venom gland. The PCR reaction was performed in 1X Taq DNA polymerase with ThermoPol (New England Biolabs, USA), 200 μM dNTPs, 0.25 μM forward primer Mlatfw (5-AGG ATA TGT TAC AAC CAA CAG - 3′); 0.25 μM reverse Mlatrv primer (5′-ACC GTT GCA TTT GTC TGA TGT -3′) and two units of Taq DNA polymerase in a final volume of 50 μL in a Applied Biosystems Gene Amp 9700 instrument. The Taq DNA polymerase was added and the mixture was then incubated at 94 °C for 3 min for one cycle. After the initial cycle, the mixture was incubated at 94 °C for 30 s, 58 °C for 2 min and 72 °C for 2 min per 30 cycles, followed by a final 7 min step at 72 °C. PCR products were purified using a High Pure PCR Product Purification Kit (Roche, Switzerland) following the manufacturer’s instructions, and then ligated into a Topo 2.1. The ligation reaction was used to transform competent E. coli XL1-Blue cells. Positive clones were sequenced from both ends using the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (Amersham, USA).
5
PCR Protocol for dNTP Synthesis
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PCR reactions (25 μL) were performed in NEB Thermopol (20 mM Tris-HCl at pH 8.8 and 25 °C with 10 mM (NH4)2SO4 10 mM KCl, 2 mM MgSO4, 0.1% Triton-X-100), 1 U NEB Taq polymerase, 0.2 μM forward and reverse primers (RSC514, RSC51532 (link); Supplementary Information, Table S1 ), template DNA (25 ng plasmid pETMCSIII-dNK; Supplementary Information, Table S2 ) and 2.5 μL of the dNTP synthesis reaction mixture to a final volume of 25 μL. The thermal treatment was 95 °C initial denaturation, 3 min; 35 cycles [95 °C denaturation, 15 s; 55 °C annealing, 30 s; 72 °C extension 30–90 s); 72 °C final extension, 7 min; then held at 4 °C until analysis by agarose gel electrophoresis. For in situ dNTP synthesis, PCR was carried out as above but with the addition of 40 mM HEPES, 0.2 mM of each deoxynucleoside, 0.2 mM ATP, 10 mM acetyl phosphate and 0.02–0.22 μL lysate from the overexpression of dNK (final PCR mixture pH 8.2) instead of the dNTP synthesis reaction mixture. A 240 min incubation at 37 °C was added to the beginning of the thermal treatment.
6
Transcriptional Analysis of Mycobacterial Efflux Pumps
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Total RNA from M. smegmatis was isolated as previously described [24 ], from bacteria grown to OD600nm 0.5. Residual DNA in the mycobacterial total RNA preparations was removed using DNase I according to the manufacturer's instructions (Sigma-Aldrich, Inc., Darmstadt, Germany). A control with no reverse transcriptase (NRT) was used for a PCR reaction with primers to 16S rRNA [25 (link)] (Table 2 ) to confirm complete DNA removal. The M-MuLV Reverse Transcriptase (New England Biolabs, MA, USA) and random primers 9 (New England Biolabs, MA, USA) (Table 2 ) were used to produce cDNA according to the manufacturer's instructions. The cDNA was used to amplify the mfp genes, in triplicate, using ThermoPol® (New England Biolabs, MA, USA) and specific primers (see Figure 1 and Table 1 : mfpED, mfpDC, mfpCB, mfpBA, and mfpA). After PCR amplification, the fragments were sequenced with the amplification primers in the forward and reverse directions (Macrogen, Korea).
7
Milk Lysate Preparation for LAMP Analysis
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In an effort to minimize costs associated with routine testing, milk lysates were prepared for analysis via LAMP by a technique modified from that previously reported (Meiri-Bendek et al., 2002) . Briefly, 1 mL of milk was placed into a 1.5-mL microfuge tube and centrifuged at 20,000 × g for 2 min in a microfuge (model Eppendorf 5424, Eppendorf South Pacific, North Ryde, NSW, Australia). Following this, the milk fat was removed from the surface of the sample with a sterile plastic loop and a sterile cotton swab and the supernatant was discarded. The sediment pellet was resuspended in 1× Tris-EDTA, pH 8.0, and centrifuged at 20,000 × g for 2 min. This step was repeated as required until the supernatant appeared clear. The sediment was resuspended in a PCR master mix 1× reaction buffer (Thermopol, New England Bio-Labs, Arundel, QLD, Australia) and lysozyme (1 μg/ μL, from chicken egg white; Sigma, Castle Hill, NSW, Australia) and incubated for 20 min at room temperature. Following this, proteinase K (5 μL of 20 mg/mL; Qiagen) was added to the suspension, which was then vortexed and incubated for 1 h at 56°C, followed by a further 15-min incubation at 100°C. The solution was then centrifuged for 1 min at 20,000 × g. The supernatant containing the milk lysate was harvested from the pellet and analyzed immediately by LAMP or stored at -20°C for later analysis.
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