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 μl
2. 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