Assemble reactions on ice. A manual hot start practice is recommended to improve specificity. Add the master mix last and start PCR cycling immediately. Add the following components to a thin-walled PCR tube on ice: COMPONENT 25 μl REACTION 50 μl REACTION FINAL CONCENTRATION Multiplex PCR 5X Master Mix 5 μl 10 μl 1X 1 μM Primer Stock 3.75 μl 7.5 μl 0.15 μM (0.05-0.4 μM) Template DNA variable variable <1,000 ng Nuclease-Free Water to 25 μl to 50 μl Note: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid. Transfer PCR tubes to a PCR machine with the block preheated to 95°C and begin the programmed cycling: STEP TEMP TIME Initial Denaturation 95°C 1 minute 30-40 Cycles 95°C 55-68°C 68°C 20 seconds 1 minute 1-2 minutes/kb* Final Extension 68°C 5 minutes Hold 4-10°C *The specified extension time is based on the longest amplicon in the reaction. For example, in a 4-plex reaction of 200, 300, 400 and 500 base pairs we recommend an extension time of 30 seconds to 1 minute at 68°C. Usage Notes: Use high quality primers (desalted or HPLC purified). Accurately quantify and adjust primer stock concentrations to 50 μM in 0.5X TE Buffer. Individually test the PCR primer pairs, preferably in a temperature-gradient PCR machine. Mix all primers equally at 1 μM in 0.5X TE buffer. Test multiplex PCR with equal molar concentration of all primer pairs, preferably in a temperature-gradient PCR machine.