Team:SDU-Denmark/labwork/Protocols/3A

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iGEM TEAM ::: SDU-DENMARK courtesy of NIAID


mRNA Isolation PCR Miniprep Check Digest
3A-Assembly Content Content Content
Content Content Content Content

3A-Assembly -Ligation of 2 parts into a vector

  1. PCR step; see PCR protocol
  2. Digestion
    3. BSA is only needed for conventional restriction enzymes, NOT fast digest
    1. Digest step for Plasmid Backbone

      2. if sample comes from miniprep
      Enzyme Master Mix for Plasmid Backbone (25ul total)
      • 5 ul Fastdigest greenbuffer / NEB Buffer 2
      • 0.5 ul BSA
      • 0.5 ul EcoRI-H
      • 0.5 ul PstI
      • 19 ul dH20 (0,5 ul less when using BSA)

      If sample comes from PCR Enzyme Master Mix for Plasmid Backbone (25ul total)
      • 5 ul Fastdigest greenbuffer / NEB Buffer
      • 0.5 ul BSA
      • 0.5 ul EcoRI-HF
      • 0.5 ul PstI
      • 0.5 ul DpnI (Used to digest any template DNA from productio
      • 19 ul dH20 (0,5 ul less when using BSA

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