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HotStarTaq PCR Handbook

1. Thaw 10x PCR Buffer, dNTP mix, primer solutions, and 25 mM MgCl2 (if required). It is important to mix the solutions completely before use to avoid localized concentrations of salts.

2. Prepare a master mix according to Table 1. The master mix typically contains all the components needed for PCR except the template DNA. Prepare a volume of master mix 10% greater than that required for the total number of PCR assays to be performed. A negative control (without template DNA) should always be included. The optimal Mg2+ concentration should be determined empirically but in most cases a concentration of 1.5 mM, as provided in the 1x PCR Buffer, will produce satisfactory results.

Table 1. Reaction composition using HotstarTaq DNA Polymerase



* Contains 15 mM MgCl2

Table 2. Final Mg2+ concentrations




3. Mix the master mix thoroughly and dispense appropriate volumes into PCR tubes. Mix gently, e.g., by pipetting the master mix up and down a few times. It is not necessary to keep PCR tubes on ice as nonspecific DNA synthesis cannot occur at room temperature due to the inactive state of HotStarTaq DNA Polymerase.

4. Add template DNA (<=1 g/reaction) to the individual tubes containing the master mix. For RT-PCR, add an aliquot from the reverse transcriptase reaction. This should not exceed 10% of the final PCR volume (see appendi
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