Mesoplasma florum: Tn5 Transposase

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Tn5 Transposase production

Tn5 transposase is the key enzyme in forming transposomes for random transposon insertions. It is sold by Epicentre at ridiculously high price. Here, we make it from a plasmid provided by Prof. Wolfgang Hillen PMID 16820464. The protocol also builds on the NEB IMPACT-CN protein purification kit.

Materials

Protocol

  • Grow BL21(DE3)pLysS cells transformed with pWH1891 in 10 ml culture.
  • Inoculate 2 liters of LB/Cm/Amp culture medium with the culture and grow overnight at 23°C
  • Induce cultures at OD 0.5 with 50 mM IPTG and grow an additional 5 hours
  • Spin down cultures and resuspend in 50 ml TEGX buffer/original liter, transferring to 50 ml centrifuge tubes
  • Spin down a second time and resuspend in 10 ml/original liter TEGX + Roche Complete protease inhibitor
  • Sonicate 3x pausing 10 minutes between sonications with the cells on ice
  • Centrifuge to remove cell debris
  • Load a 2 cm diameter column with 20 ml chitin bead suspension (10 ml beads)
  • Wash the column with 100 ml TEGX buffer
  • Load the cell supernatent onto the column and allow it to flow through
  • Flow the supernatent past the column a second time
  • Wash the column with 200 ml TEGX buffer
  • Add
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