Unless otherwise stated, all reagents used were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Thermo Fisher Scientific (Waltham, MA, USA). All the DNAs were synthesized by Integrated DNA Technologies, Inc. (IDT, Coralville, IA, USA), and the sequence information is attached to the Supplementary Materials (Table S1) . Restriction enzymes, AvrII and AvaII, and polymerases, Therminator DNA polymerase, Bsu DNA polymerase, Klenow fragment, and AMV reverse transcriptase, were purchased from New England Biolabs (Ipswich, MA, USA). Triphosphate forms of modified nucleosides were purchased from TriLink BioTechnologies (San Diego, CA, USA).
Therminator dna polymerase
Manufactured by New England Biolabs
Sourced in United States
Therminator™ DNA Polymerase is a robust and reliable enzyme designed for high-performance DNA amplification. It exhibits exceptional thermostability and can efficiently amplify DNA targets up to 20 kb in length. This polymerase demonstrates exceptional fidelity and is suitable for a wide range of PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Amv reverse transcriptase, by New England Biolabs (2 mentions)
Avaii, by New England Biolabs (1 mentions)
Klenow fragment, by New England Biolabs (1 mentions)
Vent exo dna polymerase, by New England Biolabs (1 mentions)
Lc 10ad pump, by Shimadzu (1 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
Dimethylformamide dmf, by Thermo Fisher Scientific (1 mentions)
Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
Mung bean nuclease, by New England Biolabs (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
5 protocols using therminator dna polymerase
1
Synthesis and Analysis of Modified DNAs
2
Cyclic Nucleotide Incorporation and Imaging
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After the fill-and-lock step, each sequencing cycle was performed as follows. The flow cell was incubated at 37 °C for 2 min with VT incorporation buffer (20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM NaCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 5 U/ml Therminator™ DNA polymerase (NEB) and 1 mM MgSO4 with either 125 nM VT-C, 125 nM VT-T, 200 nM VT-A, or 75 nM VT-G), followed by rinsing with Wash A and Wash B. Next, the flow cell was filled with freshly prepared imaging buffer, and fluorescence images were captured with a 200-ms exposure, after which the flow cell was rinsed with Wash A and Wash B. Subsequently, cleavage buffer and iodoacetamide buffer were respectively incubated at 37 °C for 5 min, and the flow cell was rinsed with Wash A and Wash B. Again, the imaging process, followed by washing with Wash A and Wash B was performed.
3
Enzymatic Synthesis of DNA Oligonucleotides
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All nucleotides (dAMP, dTMP, dGMP, dCMP), Dowex-50W ion exchange resin (hydrogen form), and all organic reagents were purchased from Sigma Aldrich. Dimethylformamide (DMF) was purchased from Acros Organics. Klenow fragment DNA Polymerase exo-, KlenTaq DNA Polymerase, Therminator™ DNA Polymerase, Vent® exo- DNA Polymerase, AMV Reverse Transcriptase, and dNTPs were purchased from NEB. Platinum Taq High-Fidelity DNA Polymerase and Maxima H Minus Reverse Transcriptase were purchased from Thermo Fisher Scientific. All buffers used for enzymatic experiments were provided with the enzyme. All custom oligonucleotides were purchased from IDT. Plasmids were purchased from Addgene. High-performance liquid chromatography (HPLC) was performed using a system comprised of two Shimadzu LC-10AD pumps, SCL-10A controller, and SPD-M10A photodiode array detector. Waters SunFire® C18 5 μm 4.6 × 150 mm column was used for HPLC analysis. The thermal cycler used for PCR was Eppendorf Mastercycler Gradient. PAGE analysis was visualized using a Typhoon 9410 Variable Mode Imager.
4
Mapping MazF-dr 16S rRNA Cleavage Sites
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For a primer extension analysis of the cleavage sites of MazF-dr, the 16S RNA was digested with MazF-dr at 37°C for 10 min. The reaction mixture contained 1 μg of 16S RNA substrate, 0.05 μg of MazF-dr and 0.5 μl of RNase inhibitor in 20 mM Tris-HCl buffer (pH 8.0). Primer extension was carried out in 10 μl of the reaction mixture containing 5′-FAM labeled primers (16S-F1, 16S-F2, and 16S-F3 as shown in Supplementary Table S2 ) at 42°C for 1 h, and then it was stopped by adding 2 μl of quench loading buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol EF). The samples were incubated at 70°C for 5 min prior to electrophoresis on a 6% sequencing gel. The four sequence markers were produced using PCR with 16S DNA as template and the same primers that were used in the primer extension assay. The PCR mixture contained 1 μg of 16S DNA substrate, 0.05 mM ddNTPs, 0.5 mM dNTP, therminator DNA polymerase (New England Biolabs) and 1 mM FAM labeled primer. A sequence logo plot of multiple sequence alignment was generated by the Weblogo online program to analyze the conserved RNA cleavage sites of MazF-dr2 (Doerks et al., 2002 (link)).
5
DNA Sequencing Library Preparation
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Input DNA was fragmented (total 500 ng/μg DNA; for 150–300 bp fragments, >20 000× coverage could be achieved for ends from both the positive and negative strands of the 14.2 kb plasmid). The biotin-A1 adapter was ligated to the fragmented DNA, and the ligation products were captured onto T1 streptavidin magnetic beads (Thermo Fisher, 65601). Then, the DNA on the magnetic beads was denatured with 0.1 M NaOH (Sigma−Aldrich, 79724) for 10 min, and primer was annealed for DNA strand extension. Therminator™ DNA Polymerase (NEB, M0261) was used to incorporate one 3′-hydroxyl-reversible dNTP (Jena Bioscience, 3′-O-N3-dNTPs). The 3′-hydroxyl group of the dNTPs was recovered by 100 mM TCEP (Sigma−Aldrich, 646547) treatment, and incorporation-and-reversion reactions were repeated for another 22 cycles. Then, the DNA was blunted with mung bean nuclease (NEB, M0250) for 30 min, and T4 poynucleotide kinase (NEB, M0201) was utilized to phosphorylate DNA for 30 min.
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