Polymerase Chain Reaction (PCR)

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

Procedure

  1. Prepare Master Mix 1 in a 500 µL tube on ice:
  2. Prepare Master Mix 2 in a 500 µL tube on ice:
  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:
  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

 


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