Journal: Nucleic Acids Research

Article Title: Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event

doi: 10.1093/nar/gkx171

Figure Lengend Snippet: Fe 2+ or Mn 2+ can replace Mg 2+ as a cofactor for Deep Vent (exo-) DNA polymerase. Reaction mixtures with Fe 2+ or Mg 2+ or Mn 2+ or no divalent cation were analyzed for amount of reactants and products at every other PCR cycle for 24 cycles. Reactant consumption and product yields are identical within the uncertainty of this experiment among ( A ) Fe 2+ , ( B ) Mg 2+ or ( C ) Mn 2+ . ( D ) The divalent-minus controls, which lack divalent cations, did not generate product. Neither did the ‘minus template’ negative control reactions (far left lanes of each gel), taken through 24 cycles, generate product.

Article Snippet: Polymerase chain reactions (PCR) was performed in a Coy anaerobic chamber in 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 0.1% Triton ® X-100, 100 μg/ml nuclease-free bovine serum albumin, 1 mM dNTPs, 500 nM Cy3-labeled reverse primer, 500 nM forward primer, 0.5 nM template, 1 U DeepVent ® (exo-) DNA polymerase (New England Biolabs).

Techniques: Negative Control