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- 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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