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HotStarTaq PCR Handbook

b> Prepare a master mix according to Table 3.
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.

Table 3. Reaction composition using HotStarTaq DNA polymerase and Q-Solution



* Contains 15 mM MgCl2

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 the 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. The volume added should not exceed 10% of the final PCR volume (see appendix)
.
5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 100 l mineral oil.

6. Program the thermal cycler according to the manufacturers instructions.
Each PCR program must start with an initial heat activation step at 95C for 15 min A typical PCR cycling program is outlined on
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