New England Biolabs, M0257L, Vent® (exo-) DNA Polymerase

Vent 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 0.1% Triton® X-100 50% Glycerol pH 7.4 @ 25°C Heat Inactivation No Molecular Weight Theoretical: 89000 daltons 5' - 3' Exonuclease No 3' - 5' Exonuclease No Strand Displacement +++ Unit Assay Conditions 1X ThermoPol Reaction Buffer, 200 µM each dNTP including [ 3 H]-dTTP, 15 nM primed M13 DNA. Error Rate < 190x10 -6 bases FAQ Q: Can Vent (exo-) DNA Polymerase be used in other buffers? A: Substituting other buffers is not recommended. Q: Are the DNA fragments produced by Vent (exo-) DNA Polymerase blunt-ended or do they have the single-base 3' overhang that Taq DNA Polymerase yields? A: There are predominantly two kinds of ends seen; 70% are blunt-ended, while 30% are predominantly single base 3' overhangs. If >95% blunt-ended fragments are desired, use a proofreading DNA Polymerase such as Vent DNA Polymerase (NEB# M0254). Q: I can't get Vent (exo-) 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 pair. 5. Optimize the magnesium level (2, 4, 6, or 8 mM). 6. Optimize the amount of polymerase (1-2 units per 100 μl reaction for proofreaders; 2-4 units per 100 μl reaction for exo- forms); scale up or down according to reaction volume. Details on optimization protocols may be found under the protocols tab. Q: What should I take into consideration when designing a set of PCR primers? A: 1. Avoid complementarity between the primers to prevent primer-dimer formation. 2. Avoid inverted repeats (self-complementarity). 3. GC-content of the primer should be around 50%. 4. Avoid Gs and Cs at the 3'-end of the primers. There are many computer programs which will help you to design a primer pair, such as Primer3. Q: How can I facilitate the amplification of templates with hairpin-loop structures or high GC-content? A: OneTaq® DNA Polymerase (NEB# M0480, NEB# M0481) has been developed with a GC Buffer and High GC Enhancer that aid amplifications of difficult templates. Q5® High-Fidelity DNA Polymerase is supplied with buffers and High GC Enhancer, and has been optimized for robust amplification, regardless of GC content. You could also try 7-deaza-dGTP in conjuction with normal dGTP in order to destabilize difficult structures. Note: 7-deaza-dGTP attenuates the signal of ethidium bromide staining. OneTaq® and Q5® are registered trademarks of New England Biolabs, Inc.