DNA polymerases were purchased from New England Biolabs (Vent, Vent (exo-), DeepVent (exo-), Bst 2.0, and Therminator) and Thermo Scientific (Taq, Pfu, and Phusion). The reaction mixture (25 μL) contained either 4 μM primer and template or 4 μM hairpin oligonucleotides. The dNTP concentrations were 40 or 200 μM (each) in corresponding buffer. ThermoPol® buffer (New England Biolabs) was used for the Vent, Vent (exo-), DeepVent (exo-), Bst 2.0, and Therminator polymerases. A buffer supplied by the manufacturers was used in the reactions with Taq, Pfu, and Phusion polymerases. One and a half microliters of 25 mM MgCl2were added to the reaction mixtures with Taq polymerase. Two units of enzyme were used for all polymerases, except Bst2.0 (8 units). To initiate pyrophosphorolysis, PPi was added to the reaction mixture at 0.4 mM if not otherwise stated. The reaction mixtures were heated to 95 °C for 1 min and subsequently cooled to 55 °C for 1 min. PE or hairpin modification reactions were performed at 64 or 72 °C for varying amounts of time.
Therminator
Manufactured by New England Biolabs
The Therminator is a high-performance thermal cycler designed for efficient and precise nucleic acid amplification. It features accurate temperature control and rapid heating and cooling rates to ensure reliable and reproducible PCR results.
Automatically generated - may contain errors
2 protocols using therminator
1
Comparative Evaluation of DNA Polymerases
2
Fluorescent Oligonucleotide Synthesis and Purification
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Example 1
HPLC-purified oligonucleotides and their complementary oligonucleotides were purchased from Integrated DNA Technologies (IDT). A template-dependent DNA polymerase incorporation assay was employed to incorporate a fluorescently tagged photoactivatable terminating nucleotide analog onto the 3′ end of oligonucleotide: (1) 5 μM of oligonucleotide, 25 μM of complementary oligonucleotide, 50 μM of a fluorescently tagged photoactivatable terminating nucleotide analog, 4 mM MgSO4, and 0.1 U/μL of Therminator (New England Biolabs) were mixed in 1× ThermoPol buffer, (2) the mix was heated to 80° C. for 45 seconds, and (3) the mix was incubated for 5 minutes at each of 60° C., 55° C., 50° C., 45° C., 40° C., 35° C., 30° C., and 2555° C. The incorporation product was purified on the Agilent 1260 Infinity reverse phase HPLC using the XTerra MS C18 Prep column (Waters). The purified product solution volume was concentrated to approximately 250 μL using the Eppendorf Vacufuge followed by denaturation into single-stranded oligonucleotides with an equal volume of 0.2 M NaOH. HPLC purification and concentration were repeated using the same conditions for collection of the oligonucleotides. The final product was dissolved into 1×PBS, and concentration was determined by measuring fluorescent dye absorbance.
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