Mesoplasma florum:Transposome construction

• Cut the transposome out of the containing plasmid with PvuII enzyme, which cuts at the correct location at the mosaic end.
• Cut also with an enzyme which produces shorter fragments from the remaining plasmid backbone to make gel purification easier. Sau3AI is a good enzyme.
  • Use NEB Buffer 1 with BSA which is good for both Sau3AI and PvuII.
• TT01 transposon is 2478 bp long
• Reaction
  • 20 μg of plasmid DNA from a maxiprep
  • 10 μl NEB Buffer 1
  • 1 μl 100x BSA
  • 3 μl PvuII
  • 1 μl Sau3AI
  • QS water to 100 μl
  • Heat 37° 1 hour
  • Heat kill the enzymes 20 minutes at 65°
  • Add 20 μl loading dye
  • Load and run on a prep gel
  • Cut the band at 2478 bp from the gel
  • Weigh the cut band
  • Add 3x volume Qiagen QX1 buffer
  • Add 30 μl Qiaex II suspension
  • Heat at 50° while vortexing until completely dissolved
  • Spin, discard, resuspend in 1 ml QX1 buffer
  • Spin, discard, resuspend in 1 ml PE buffer
  • Spin, discard, resuspend in 1 ml PE buffer
  • Spin, discard, spin again, remove remaining PE buffer with 10 μl tip
  • Dry at 50° for 15 minutes until the Qiaex II turns white
  • Resuspend in 30 μl TE
  • Spin, remove supernatent to a fresh tube with a 10 μl tip
  • Add an additional 10 μl TE to the Qiaex II suspension, resuspend, spin
  • Remove supernatent with a 10 μl tip
  • Spin down the fresh tube to pellet any remaining Qiaex II suspension and transfer supernatent to a screw to vial
  • Measure concentration and label the tube