Amplitaq Gold

Enzyme 1.5 U AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA). 

A real-time PCR master-mix was made consisting of 1.25 pL of the forward and reverse primers, 5pL Amplitaq buffer, 4pL MgCl2,2.5 pL of dNTPs, 3.75 pL of TaqMan probe, 1 pL of the ROX reference dye, 26pL of ddH20, and 0.3 pL of Amplitaq gold polymerase. 

A 20-pL PCR is ideal for Pyrosequencing however, if the PCR product is especially strong or wide-peak pyrograms occur, a 10-pL reaction will work well. For a 20-pL reaction based on ABl Amplitaq Gold PCR mastermix (Applied Biosystems, Foster City, CA) ... 

PCR is performed in a total volume of 25 juL. The reaction mixture contains 0.2 mM of each deoxynucleotide triphosphate, 0.2 pM of each primer, 1 x PCR buffer (GeneAmp PCR buffer. Applied Biosystems), 2 mM MgCl2,1.5 units Taq polymerase (AmpliTaq Gold DNA Polymerase, Applied Biosystems), and 2 pL (30 to 100 ng) of template DNA solution. Primers and PCR conditions are listed in Tables 7.1 to 7.3. 

As an alternative to manual Hot Start, AmpliTaq Gold DNA polymerase (250 units, PE Applied Bio stems, N808-0241) can be used without having to re-optimize the PCR conditions. 

Applied Biosystems is a trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries. GeneAmp and AmpliTaq Gold are registered trademarks of Roche Molecular Systems, Inc. 

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