Fluorescent primer extension was performed in 50 μl reaction volumes using the entire footprinting reaction product. Each reaction contained 1x ThermoPol Buffer (New England Biolabs), 0.1 mM dNTPs, 40 nM 5’ 6-FAM labeled primer and 1 U VentR (exo-) DNA polymerase (New England Biolabs). The pagC samples were analyzed using the primer 6FAM-pagC. Reactions were denatured for 3 min at 95° C followed by 70 cycles of 1 min at 95° C, 1 min annealing at 51° C, and 1 min extension at 72° C. Reactions were purified using SigmaSpin sequencing clean-up columns (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions, dried, and dissolved in 20 μl H2O. Reactions were analyzed on an Applied Biosystems 3730xl DNA analyzer at the Fred Hutchinson Cancer Research Center Genetic Analysis Lab (Seattle, WA).
Ventr exo dna polymerase
Manufactured by New England Biolabs
VentR (exo-) DNA polymerase is a thermostable DNA polymerase with 5' to 3' polymerase and 3' to 5' exonuclease activities. It is used for high-fidelity DNA amplification.
Automatically generated - may contain errors
2 protocols using ventr exo dna polymerase
1
Fluorescent Primer Extension Analysis
2
Fluorescent Primer Extension Analysis
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Fluorescent primer extension was performed in 50 μl reaction volumes using the entire footprinting reaction product. Each reaction contained 1x ThermoPol Buffer (New England Biolabs), 0.1 mM dNTPs, 40 nM 5’ 6-FAM labeled primer and 1 U VentR (exo-) DNA polymerase (New England Biolabs). The pagC samples were analyzed using the primer 6FAM-pagC. Reactions were denatured for 3 min at 95° C followed by 70 cycles of 1 min at 95° C, 1 min annealing at 51° C, and 1 min extension at 72° C. Reactions were purified using SigmaSpin sequencing clean-up columns (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions, dried, and dissolved in 20 μl H2O. Reactions were analyzed on an Applied Biosystems 3730xl DNA analyzer at the Fred Hutchinson Cancer Research Center Genetic Analysis Lab (Seattle, WA).
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