Hi, I am facing a problem in screening the normal silk gene (476 bp) in wild type and Cas9 mutant samples (Cas9 targeting the 476 bp gene) using high fidelity polymerases in the PCR. As you can see on the left side image of the picture, I used the high fidelity (mixture of polymerase; Taq and pfu) polymerase to amplify the silk gene of 476 bp but only the wild-type (outbreed) produced the amplification. However, when I used only the Taq polymerase, it yielded the 476 bp genes both in the wild-type (outbreed) and the mutant samples (right hand side image). The concentration of gDNA for all samples used in the PCR were relatively the same about 9-10 ng/ul. Does anyone has explanation to this?
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