IGEM:Paris Bettencourt 2012/Protocols/RCR Taq

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  1. Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
  2. Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
  3. PCR Master Mix (2x)
    25 ul
    Forward primer
    0.1 - 1.0 uM
    Reverse primer
    0.1 - 1.0 uM
    Template DNA
    10 pg - 1 ug
    Water (Nuclease free)
    to 50 ul
    Total volume:
    50 ul
  4. Gently vortex the samples and spin down.
  5. When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
  6. Perform PCR using the recommended thermal cycling conditions outlined below:
  7. Step Temperature, °C Time Number of cycles
    Initial denaturation
    95
    10 min
    1
    Denaturation
    95
    30 s
    30
    Annealing
    50
    30 s
    Extension
    72
    1 min/kb
    Final Extension
    72
    10 min
    1
    End
    8
    Forever
    1


  • Comments: LID = 100°C
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