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