This procedure is a "hot start" PCR cycle for use with large primers. PCR mass-produces DNA contained in the source plasmid between the two primer-binding sequences, eventually producing linear DNA starting with Primer 1, continuing with the source sequence, and ending with the complementary sequence to Primer 2.

Materials

• 10 mM dNTP mix [Boehringer Mannheim, cat # 1581 295]
• 10X PCR Buffer w/ 15 mM MgCl2 [Boehringer Mannheim, cat # 1759 167]
• PCR Enzyme: Expand HF PCR System [Boehringer Mannheim, cat # 1732 641]
• 150 µM Primer 1
• 150 µM Primer 2
• Source plasmid
• Mineral oil
• Chloroform: ACS Reagent Grade
• PCR Thermocycler

Procedure

1. Prepare Master Mix 1 in a 500 µL tube on ice:
   • 2 µL dNTP solution
   • 2 µL dilute Primer 1 (dilute 1:10 in sterile dH2O)
   • 2 µL dilute Primer 2 (dilute 1:10 in sterile dH2O)
   • 4 µL source plasmid (adjust amount as necessary)
   • 40 µL sterile dH2O
2. Prepare Master Mix 2 in a 500 µL tube on ice:
   • 10 µL 10X PCR Buffer
   • 0.75 µL PCR Enzyme
   • 39 µL sterile dH2O
3. Immediately before starting the PCR cycle, add MM2 to MM1; to prevent evaporation, place two drops of mineral oil on top of the solution
4. Run the following PCR cycle:
   • First cycle: 5 minutes at 95 °C
   • Next thirty cycles: 1 minute at 94 °C, 2 minutes at 55°C, 3 minutes at 72 °C
   • Hold at 4 °C
5. Add 150 µL of Chloroform to the PCR product to remove mineral oil; gently invert to mix
6. Transfer the aqueous "bubble" (floating on top) to a fresh 500 µL tube with a gel-loading tip on a 20 µL micropipet
7. Store PCR Product at -20 °C