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Revision as of 12:32, 9 September 2015

Analytical digestion protocol

  • Prepare the following mix:
    • 2µL 10X Digestion buffer
    • 0.5µL Eco31I
    • 0.5µL BbsI
    • 2µL of DNA (200ng)
    • 15µL water
  • incubate 1h at 37°C

Annealing Protocol

  • Phosphorylation of the oligos
    • 5.6μL DNAse/RNAse free water
    • 6.0μL oligo 1 (10µM)
    • 6.0μL oligo 2 (10µM)
    • 2.0μL 10X T4 DNA ligase buffer
    • 0.4μL T4 PolyNucleotide Kinase
      • Total: 20μL
  • incubate 30min at 37°C
  • add 1μL of 1M NaCl
  • incube 5min at 95°C
  • let the mix cool down
  • use 2μL of the mix as a 10X solution

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