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TripleMaster PCR System

primer mix containing DMSO (see step 4 in protocol below and table 8). Protocol for GC-rich targets
  1. Thaw the reagents listed in Table 8 carefully and keep on ice.
  2. Make sure reagents are thoroughly thawed. Mix each reagent and briefly centrifuge before use.
  3. Set up Mastermix 1 (see Table 8) containing buffer, primer, template DNA and DMSO (optional) in a sterile microcentrifuge tube, mix well, centrifuge and heat for 30 seconds at 98C.
  4. Quickly spin down (2 sec) Mastermix 1 and immediately place on ice.
  5. Prepare Mastermix 2 (see Table 8) in a separate sterile microcentrifuge tube, mix well, centrifuge and place on ice.
  6. Just before cycling, pipette Mastermix 1 and then Mastermix 2 into a separate PCR tube on ice. Mix quickly but thoroughly, (vortex) and centrifuge.
  7. Place immediately into a thermal cycler that is pre-heated at 98C (use the "Hold" step for this) and start cycling.
  8. Analyze amplification products on a 0.6-1.5 % agarose gel using appropriate DNA markers.
Table 8: Mastermix conditions for GC-rich PCR 1 increase final buffer conc. (1.6x) for targets < 500 bp or when using a low concentration of template DNA per reaction (< 10 ng genomic DNA or < 0.5 ng plasmid DNA)
2 increase dNTP concentration for GC-rich targets

Table 9: Cycling program for GC-rich targets 1 Difficult targets require more cycles (and template DNA) for efficient amplificati
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