Mesoplasma florum:Inverse PCR Transposon location

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Materials

  • Genomic DNA from single colony transposon insertion event
  • MboI
  • NEB buffer 3 10x
  • T4 DNA ligase buffer 10x
  • T4 DNA ligase
  • PCR supermix
  • M13forward(-47) primer
  • T7 universal primer
  • ME primer
  • E-Gel 0.8%

Restriction digest of 500 ng of genomic DNA with MboI

  • Mix 0.5 μl (approx) genomic DNA in TE
  • 5 μl NEB buffer 3
  • 1 μl MboI
  • 43.5 μl water
  • incubate at 37° for 30 minutes
  • heat kill at 65° for 20 minutes

Ligation of cut ends at 5 ng/μl concentration (Hutchison99)

  • 5 μl digested DNA
  • 1 μl T4 DNA ligase buffer
  • 0.2 μl T4 DNA ligase
  • 3.8 μl water
  • incubate 10 minutes at room temperature

Transposon detection PCR reaction 10 μl test volume

  • 0.5 μl ligated DNA
  • 0.3 μl ME primer

9.2 μl PCR supermix High Fidelity

Inverse PCR for transposon location identification

  • 0.5 μl ligated DNA
  • 0.15 μl M13forward(-47) primer
  • 0.15 μl T7 universal primer
  • 9.2 μl PCR supermix high fidelity
  • Cycle 5 minutes at 95° initial denturation
  • 40 cycles of
    • 94° 30 seconds
    • 55° 30 seconds
    • 64° 3 minutes
  • 10 minutes 64° final extension

Detect with E-Gel 0.8%

Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650

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