( avoid repeated freezing and thaw...)QIAGEN Multiplex PCR Handbook

avoid repeated freezing and thawing. Multiple freeze/thaw cycles of the primer mix may lead to decreased assay performance.

Table 1. Preparation of 10x primer mix (containing 2 M each primer)*

* Allows preparation of a 10x primer mix containing up to 12 primer pairs (50 M stock) or containing up to 25 primer pairs (100 M stock).

Standard Multiplex PCR Protocol

Important notes before starting

• Always start with the cycling conditions specified in this protocol.
  
• If using an already established multiplex PCR system, use the previously established annealing temperature in combination with the cycling conditions specified in this protocol.
  
• Annealing time must be 90 s.
  
• Use equal concentrations (0.2 M) of all primers.
  
• PCR must start with an activation step of 15 min at 95C to activate HotStarTaq DNA Polymerase (see step 7 of this protocol).
  
• Optional: If a thermal cycler with a temperature gradient function can be used, determine the optimal annealing temperature by performing a gradient PCR.

Protocol

1. Thaw 2x QIAGEN Multiplex PCR Master Mix (if stored at 20C), template DNA, distilled water, and primer mix. Mix the solutions completely before use.
It is important to mix the solutions completely before use to avoid localized concentrations of salts. Preparing a mixture of all primers avoids pipetting of individual primers for each experiment, reducing
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