Mesoplasma florum:Transposome construction

Transposome DNA construction from plasmid cutting

• 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

Transposome DNA construction using PCR and cutting

• PCR with ext-ME primer using Phusion master mix. Final volume 200 μl
  • 100 μl 2x Phusion master mix
  • 6 μl ext-ME primer (30 pM/μl) (gt ttc ttc agc tgt ctc tta tac aca tct)
  • 1 μl transposon template (10 ng/μl)
  • 93 μl water
  • Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
• Add 1 ml Qiagen buffer PB
• Bind to Qiaquick column 2x 600 μl, flowing past the column twice
• Wash 2x with 750μl buffer PE, spin after emptying.
• Elute with 50 μl buffer EB into a clean tube.
• Add 10 μl NEB buffer 2
• Add 37 μl water
• Add 3 μl PvuII, mix
• Incubate at 37° for 1 hour
• Add 300 μl Qiagen buffer ERC
• Bind to a Qiagen Minelute column by flowing 2x through the column
• Wash 2x with 750 μl buffer PE
• Spin dry
• Elute with 20 μl TE
• Measure concentration of the resulting DNA

Transposome DNA construction using PCR

• PCR with ME0 primer using Phusion master mix. Final volume 200 μl
  • 100 μl 2x Phusion master mix
  • 6 μl ME0 primer (30 pM/μl) (phos- c tgt ctc tta tac aca tct)
  • 1 μl transposon template (10 ng/μl)
  • 93 μl water
  • Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
• Add 1 ml Qiagen buffer PB
• Bind to standard Qiagen column, flowing past the column twice
• Wash 2x with 750μl buffer PE, spin after emptying.
• Elute with 100 and 50 μl TE
• vacuum evaporate, resuspend in 10 μl TE
• Measure concentration of the resulting DNA, expect 1 μg/μl
• Dilute 0.5 μl into 20 μl, run a gel to verify correct size (TT01 is 2478 bp)

Final transposome construction

• 1 μL transposon DNA, with phosphorylated ME ends, 100 ng/μL
• 2 μL EZ-Transposase (Epicentre) [1]
• 1 μL glycerol
• hold at RT for 1/2 hour
• reported to age at 4C overnight to provide higher efficiency