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HotStarTaq PCR Handbook

br> This is recommended if several amplification reactions using the same primer pair are to be performed. The final volume of diluted primer mix should be 25 l per reaction including the template DNA, added at step 4.

2. Mix the HotStarTaq Master Mix by vortexing briefly and dispense 25 l into each PCR tube according to Table 4.
It is important to mix the HotStarTaq Master Mix before use in order to avoid localized concentrations of salt. HotStarTaq Master Mix is provided as a 2x concentrate (i.e., a 25 l volume of the HotStarTaq Master Mix is required for amplification reactions with a final volume of 50 l). For volumes smaller than 50 l, the 1:1 ratio of HotStarTaq Master Mix to diluted primer mix and template should be maintained as defined in Table 4. A negative control (without template DNA) should always be included. 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.

3. Distribute the appropriate volume of diluted primer mix into the PCR tubes containing the Master Mix.

4. Add template DNA (1g/reaction) to the individual PCR tubes.
For RT-PCR, add an aliquot from the reverse transcription reaction. The volume added should not exceed 10% of the final PCR volume (see appendix).

Table 4. Reaction composition using HotStarTaq Master Mix


*Contains 1.5 mM MgCl2

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