Overlap extension PCR

  1. This type of PCR is used to make mutations, fuse two gene segments together, make insertions within a gene, or make deletions within a gene.

  2. Set up the two half reactions as follows:

    Vent Polymerase Reactions for the two halves:
    • 80.5 ul distilled H2O
    • 10 ul ThermoPol buffer
    • 2 ul 10 mM dNTPs
    • 2 ul 100 mM MgSO4
    • 2 pmoles primerA or B, usually 2 ul of a 100 pM stock
    • 2 pmoles primerC or D, usually 2 ul of a 100 pM stock
    • 1 ul miniprep or Qiagen DNA, usually around 200-500 ng template
    • 0.5 ml Vent DNA Polymerase

    Note: Volume of distilled H2O can be adjusted if additional volumes of reagents are required.

    PCR Cycle (for PE 9600 or equivalent):
    95oC for 5 minutes

    95oC for 30 seconds
    55oC for 1 minute
    72oC for 2 minutes
      ==> 35 cycles

    72oC for 10 minutes
    4oC hold

    Note: The incubation times at each temperature can be varied to accommodate different volumes of reactions.

    Also, annealing temperature may have to be determined emperically.
  1. Run 50 ul PCR products on a gel.

  2. Cut out gel slices containing appropriate DNA fragments.

  3. Purify DNA from gel by squeezing gel through a glass wool filter.

  4. Set up the overlap extension PCR reaction as follows:

    Vent Polymerase Reactions for the two halves:
    • 71.5 ul distilled H2O
    • 10 ul ThermoPol buffer
    • 2 ul 10 mM dNTPs
    • 2 ul 100 mM MgSO4
    • 2 pmoles primerA, usually 2 ul of a 100 pM stock
    • 2 pmoles primerD, usually 2 ul of a 100 pM stock
    • 5 ul PCR fragment AC
    • 5 ul PCR fragment BD
    • 0.5 ml Vent DNA Polymerase

    Note: Volume of distilled H2O can be adjusted if additional volumes of reagents are required.

    PCR Cycle (for PE 9600 or equivalent):

    95oC for 5 minutes

    95oC for 30 seconds
    55oC for 1 minute
    72oC for 2 minutes
      ==> 35 cycles

    72oC for 10 minutes
    4oC hold

    Note: The incubation times at each temperature can be varied to accommodate different volumes of reactions.

    Also, annealing temperature may have to be determined emperically.
  1. Check 10 ul reactions on gel to make sure overlap extension PCR worked.

  2. Wash PCR reactions 2x with 300 ul distilled H2O in a Millipore Microcon 100 device.

  3. Elute with 50 ul TE.

  4. Digestions can now be performed using this purified sample.