User:Tk/Notebook/MF/2009/11/24

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16S PCR and random cloning of fragments

  • Dilute L1, GF, PP, Ent DNA to 10 ng/ul in TE


  • PCR reaction master mix
    • 100 μl Qiagen Syber green m/m
    • 3 μl each of primers for ends of RNA gene (1.5Kb)
    • 94 μl water
  • aliquot 50 μl add 1 μl of diluted DNA
  • Cycle
    • 95 2:00
    • 95 :30
    • 55 :30
    • 68 1:40 X37

Cut pUC19 DNA with EcoRI

  • 50 μl reaction with 1 μg DNA
    • 30 min/heat kill

Phosphatase treat 1/2 of the reaction

  • Aliquot and add 2.5 μl Antarctic phosphatase buffer to 25 μl, add 1 μl antarctic phosphatase
    • 30 min/heat kill

Ligate with EcoRI digests from 11/21/09

  • Master mixes of 1 μl cut pUC19, 1 μl T4 ligase, buffer
  • Add 2 μl EcoRI cut DNA
  • ligate at RT 20 minutes, then 20 min at 4C

Transform onto XGal + IPTG plates (actually S-Gal)