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For nested PCR, a final concentration 1X Q5 Buffer, 1X GC enhancer, 0.25 mM dNTPs, Q5 enzyme (1:50), water as well as 0.2 M of cocktail of nested forward primers and 0.2 M of a cocktail of nested reverse primers were mixed with 25 L of purified linked product template to generate as many 200 L reaction as needed to amplify all of the linked template

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