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All PCR experiments should be assembled in a nuclease-free environment. In addition, DNA sample preparation, reaction set-up, and subsequent reaction(s) should be performed in separate areas to avoid cross contamination. The use of "clean" pipettors designated for PCR and aerosol resistant barrier tips are recommended. A negative control reaction (omitting DNA template) should be performed in tandem with sample PCR to confirm the absence of DNA contamination.1. Add the following components into a sterile 0.2 ml PCR tube on ice (50 μlreaction volume). - 2X PCR MasterMix 25 μl (Final Concentration 1X) - DNA Template ~100 ng (Final Concentration ~2 ng/μl) - Forward Primer (10 μM) 1-2.5 μl (Final Concentration 200-500 nM) - Reverse Primer (10 μM) 1-2.5 μl (Final Concentration 200-500 nM) - Nuclease-free H2O Up to 50 μl2. Mix tube contents carefully and thoroughly, then collect all reagents by a brief centrifugation.3. Choose an appropriate PCR amplification protocol for different DNA Polymerases.4. Final PCR products are analyzed by agarose gel electrophoresis