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 1 ml of 1M IPTG to 20 ml of TEGX buffer
  • Flow the 20 ml IPTG + TEGX into the column
  • Cap the column and hold at 4°C overnight
  • Recover the protein with 10 ml TEGX buffer flowed through the column


Molarity

  • Gel runs at 55 KD, approximately correct for a 450 AA protein.
  • Kostner06 uses 100-500 fmol DNA + 5x excess protein, or .5 - 2.5 pmol
  • 1 pmol protein = 55000 g/mol * 1e-12 mol = 55 ng
  • 1 pmol transposon = 2700 * 660 * 1e-12 = 1.8 ug
  • 100 fmol transposon = 180 ng


DNA binding tests

  • Use constant 200 ng of DNA (dilute from 1 ug/ul stock)
  • Use serial dilutions of protein starting at 8 ug/ul
    • 2x dilutions 0, 2, 1, 500, 250, 125, 62.5, 31.25, 15.6, 0 into 20ul TEGX + 200 ng/ul transposon DNA
  • Incubate 30 minutes at 37
  • Run on 0.8% E-Gel
  • DNA mixture 10*200 ng = 2 ug DNA = 2 ul DNA into 200 ul TEGX


Gel images, Goryshin00

  • Fig 1, 1.8 Kb transposon reacted at 2.5 ng/ul with Tn5 transposase at 10 ng/ul in 400 ul final volume, 1 hour at 37C
    • this is 181 fmol/ul of DNA and 5.5 pmol/ul transposase, or a 30x molar excess of transposase
  • Concentrated to 20 ul and run on a 1.2% gel

References

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