PickMutant™ Random Mutagenesis Kit

PCR is routinely performed with standard Polymerases, yielding a final PCR product with few errors. Although Polymerases vary in fidelity, the inherent Mutation rate of PCR is insufficient to generate the high frequency of mutations necessary to generate a mutant library. The addition of nucleotide analogues to the PCR reaction reduces Polymerase fidelity increased substitution frequency in the PCR products. PickMutant™ Random Mutagenesis (by Nucleotide Analog dPTP) Kit is based on the incorporation of Mutagenic dPTP, into an amplified DNA fragment by PCR. The Mutagenic dPTPs are eliminated by a second PCR step in the presence of the four natural dNTPs only, resulting in a rate of Mutagenesis of up to 20% (Zaccolo et. Al). dPTP is an excellent substrate for Taq Polymerase (Km =22 mM versus Km = 9. 5 mM for TTP); it is incorporated in place of TTP and, with a ≈fourfold lower efficiency, in place of dCTP. dPTP produces four transitions (A→G, T→C, G→A and C→T). Two transitions (A→G and T→C) occur at higher frequency than the other two (G→A and C→T) (Zaccolo et. Al).

Includes for 15 rxn:
– 40 μL Taq Polymerase (5 U/μL)
– 200 μL PCR Buffer (10x)
– 100 μL dNTP Mix (10 mM)
– 100 μL MgCl2 (25 mM)
– 40 μL dPTP (10 mM)
– 2 x 1 mL PCR-grade Water

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