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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