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.
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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
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 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 Immediately before starting the PCR cycle, add MM2 to MM1; to prevent evaporation, place two drops of mineral oil on top of the solution 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 Add 150 µL of Chloroform to the PCR product to remove mineral oil; gently invert to mix Transfer the aqueous "bubble" (floating on top) to a fresh 500 µL tube with a gel-loading tip on a 20 µL micropipet Store PCR Product at -20 °C
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