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Mesoplasma florum:Transposome construction
蚂蚁淘
/发布时间:1629221402 /浏览量:417
- 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
- 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 2x 500 μl Qiagen buffer PB
- Bind to Qiaquick column 2x 600 μl, flowing past the column twice
- Wash once with 500 μl buffer PB
- Wash 2x with 750μl buffer PE, spin at high speed for 5 minutes after emptying.
- Elute with 2x 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
- PCR with ME0 primer using Phusion master mix. Final volume 200 μl, split 2x 100μl
- 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 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 750μl buffer PE, spin after emptying.
- Elute with 2x 50 μl TE
- vacuum evaporate, resuspend in 20 μl TE
- Measure concentration of the resulting DNA, expect 500 ng/μl
- Dilute to 100 ng/μl
- Dilute 1 μl into 20 μl, run a gel to verify correct size (TT01 is 2478 bp)
- 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
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