( dd template DNA (= 1 g/50 l reaction) ...)QIAGEN Multiplex PCR Handbook

dd template DNA (<= 1 g/50 l reaction) to the individual PCR tubes containing the reaction mix.
For multiplex RT-PCR, the volume of cDNA added (from the RT reaction) as template should not exceed 10% of the final PCR volume. 5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 50 l mineral oil. 6. Program the thermal cycler according to the manufacturers instructions.
Optional: If a thermal cycler with a temperature gradient function can be used, determine the optimal annealing temperature by performing a gradient PCR. 7. Place the PCR tubes in the thermal cycler and start the cycling program as outlined in Table 5.
Each PCR program must start with an initial heat-activation step at 95C for 15 min to activate HotStarTaq DNA Polymerase.
After amplification, samples can be stored overnight at 28C or at 20C for longer storage. Table 5. Microsatellite cycling protocol

* Tm determined according to the formula: Tm = 2C x (number of [A+T]) + 4C x (number of [G+C]).
For targets longer than 0.5 kb, an extension time of 90 s may improve performance. 8. Analyze samples using an appropriate detection system, for example automatic gel-based DNA sequencers or those based on capillary electrophoresis.
The optimal amount of PCR product to load, to give a satisfac
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