Primer For Sequencing Prefer Tm: 50-60C, GC content: 40-60%

E Coli Colonies

  • Colonies need to grow for at least 16hrs at 37C to reach good visible size
  • Pick a single colony with sterile tip and resuspend in 30ul sterile Tris buffer (10mM, ph8.0)
  • Prepare 2 separate tubes, 1 contains 15ul colony suspension, 1 contains 5ul primer at 5pmol/ul

Plasmid Walking

  • 10ug plasmid DNA
  • Vector information if whole plasmid sequencing to be done
  • 10ul vector primers for first round of sequencing at 5uM
  • Reference or expected sequence if any