New England Biolabs, M0258S, Deep Vent® DNA Polymerase

Related Categories Other PCR Polymerases Applications Routine PCR,, PCR Specification Unit Definition One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 75°C. Reaction Conditions 1X ThermoPol® Reaction Buffer 1X ThermoPol® Reaction Buffer 20 mM Tris-HCl 10 mM (NH4)2SO4 10 mM KCl 2 mM MgSO4 0.1% Triton® X-100 (pH 8.8 @ 25°C) Storage Buffer 10 mM Tris-HCl 100 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% Triton® X-100 pH 7.4 @ 25°C Heat Inactivation No Molecular Weight Theoretical: 89000 daltons 5' - 3' Exonuclease No 3' - 5' Exonuclease Yes Strand Displacement ++ Unit Assay Conditions 1X ThermoPol Reaction Buffer, 200 µM each dNTP including [ 3 H]-dTTP and 15 nM primed M13 DNA. FAQ Q: Can Deep Vent DNA Polymerase be used in other buffers? A: Substituting other buffers is not recommended. Q: I can't get Deep Vent DNA Polymerase to work yet Taq DNA Polymerase works fine. A: DNA polymerases possessing high fidelity due to a proofreading exonuclease moiety are more exacting in their use than their exo- derivatives or DNA polymerases without a proofreading exonuclease, such as Taq DNA Polymerase. Also, differences in the Km (DNA) possessed by different DNA polymerases can change the effective primer annealing temperature. We suggest reviewing the general guidelines accompanying the polymerase and adhering to the following suggestions: 1. Use the buffer supplied with the polymerase. 2. Use the four standard dNTPs (not dUTP or dITP). 3. Some suppliers of dNTPs sell stocks contaminated with dUTP which will cause problems with our thermostable DNA polymerases. To avoid contamination, we suggest NEB dNTPs. 4. Optimize the annealing temperature for the primer. 5. Optimize the magnesium level (2, 4, 6, or 8 mM). 6. Optimize the amount of polymerase (0.25-2 units per reaction for proofreaders; 2-4 units per reaction for exo- forms); scale up or down according to reaction volume. Details on optimization protocols may be found under the general Polymerase Technical Reference page. Q: Are the DNA fragments produced by Deep Vent DNA Polymerase blunt-ended or do they have the single-base 3' overhang that Taq DNA Polymerase yields? A: Deep Vent DNA Polymerase generates > 95% blunt-ended fragments. The exo- derivatives are more like Taq DNA Polymerase in that there are predominantly two kinds of ends seen; 70% are blunt-ended, while 30% are predominantly single base 3' overhangs. Q: Can I use Deep Vent DNA Polymerase to polish the ends of DNA fragments? A: Yes, DNA polymerases with proofreading activity (3'→5' exonuclease) can be used to fill in a 5' overhang and remove a 3' extension. DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) is preferred because it works at 25°C, can be used in a variety of buffers, and can be heat killed. Q: I want to clone primer extension products made with Vent DNA Polymerase or Deep Vent DNA Polymerase. Are some protocols better than others? A: The best protocols depend on blunt-end cloning into a phosphatase-treated vector. The insert should be kinased at its 5' ends unless the primer has been synthesized with a 5' phosphate. Q: Can Vent® DNA Polymerase (NEB# M0254) or Deep Vent® DNA Polymerase (NEB# M0258) be used to create blunt ends on primer extension products made with Taq DNA Polymerase (NEB# M0267)? A: Yes. Phenol:chloroform extract and precipitate the DNA with 2 volumes of isopropanol. Resuspend in 1X ThermoPol Reaction Buffer, add 1-2 units of Vent DNA Polymerase or Deep Vent DNA Polymerase per 100 µl reaction, and 200 µM dNTPs; mix well and incubate at 72°C for 20 minutes.