Team:Groningen/protocols/PCR

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PCR

Materials:
  • PCR tubes
  • MQ water
  • DMSO 5%
  • Phusion buffer
  • DNTPs
  • F- and R-primer
  • Phusion Polymerase
  • DNA template


PCR reaction mixture:
Component 50 ml Final concentration
    MQ water
    up to 50 µl
      5x Phusion Buffer
      10 µl
      1x
      DMSO 5%
      1.5 µl
        10 mM dNTPs
        1 µl
        200 µM
        primer F
        2.5 µl
        0.5 µM
        primer R
        2.5 µl
        0.5 µM
        template DNA
        1 µl
        ∼1 ng
        Phusion Polymerase
        2 U/µl
        0.3 µl
        0.01 U/µl


      Cycling instructions:
      Temperature Time Number of Cycles
        98°C
        2 min
        1
        98°C
        10 sec
        34
        Tm-5°C
        25 sec
        72°C
        30 sec/kb
        72°C
        10 min
        1
        4°C
        ∞
        1

      Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/f-530s-product-information.pdf