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 &mu;g of plasmid DNA from a maxiprep
 * 10 &mu;l NEB Buffer 1
 * 1 &mu;l 100x BSA
 * 3 &mu;l PvuII
 * 1 &mu;l Sau3AI
 * QS water to 100 &mu;l
 * Heat 37&deg; 1 hour
 * Heat kill the enzymes 20 minutes at 65&deg;
 * Add 20 &mu;l 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 &mu;l Qiaex II suspension
 * Heat at 50&deg; 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 &mu;l tip
 * Dry at 50&deg; for 15 minutes until the Qiaex II turns white
 * Resuspend in 30 &mu;l TE
 * Spin, remove supernatent to a fresh tube with a 10 &mu;l tip
 * Add an additional 10 &mu;l TE to the Qiaex II suspension, resuspend, spin
 * Remove supernatent with a 10 &mu;l 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 &mu;l
 * 100 &mu;l 2x Phusion master mix
 * 6 &mu;l ext-ME primer (30 pM/&mu;l) (gt ttc ttc agc tgt ctc tta tac aca tct)
 * 1 &mu;l transposon template (10 ng/&mu;l)
 * 93 &mu;l water
 * Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
 * Add 2x 500 μl Qiagen buffer PB
 * Bind to Qiaquick column 2x 600 &mu;l, flowing past the column twice
 * Wash once with 500 μl buffer PB
 * Wash 2x with 750&mu;l buffer PE, spin at high speed for 5 minutes after emptying.
 * Elute with 2x 50 &mu;l buffer EB into a clean tube.
 * Add 10 &mu;l NEB buffer 2
 * Add 37 &mu;l water
 * Add 3 &mu;l PvuII, mix
 * Incubate at 37&deg; for 1 hour
 * Add 300 &mu;l Qiagen buffer ERC
 * Bind to a Qiagen Minelute column by flowing 2x through the column
 * Wash 2x with 750 &mu;l buffer PE
 * Spin dry
 * Elute with 20 &mu;l TE
 * Measure concentration of the resulting DNA

Transposome DNA construction using PCR

 * PCR with ME0 primer using Phusion master mix. Final volume 200 &mu;l, split 2x 100μl
 * 100 &mu;l 2x Phusion master mix
 * 6 &mu;l ME0 primer (30 pM/&mu;l) (phos- c tgt ctc tta tac aca tct)
 * 1 &mu;l transposon template (10 ng/&mu;l)
 * 93 &mu;l water
 * Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
 * Add 2x 500 μl Qiagen buffer PB
 * Bind to standard Qiagen column, flowing past the column twice
 * Wash once with 500 μl buffer PB
 * Wash 2x with 750&mu;l buffer PE, spin after emptying.
 * Elute with 2x 50 &mu;l TE
 * vacuum evaporate, resuspend in 20 &mu;l TE
 * Measure concentration of the resulting DNA, expect 500 ng/&mu;l
 * Dilute to 100 ng/&mu;l
 * Dilute 1 &mu;l into 20 &mu;l, 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 μL glycerol
 * hold at RT for 1/2 hour
 * reported to age at 4C overnight to provide higher efficiency