《Science》发表非损伤微测技术研究Ca2+流速的成果分析方法生物...相关交流-蚂蚁淘在线

《Science》发表非损伤微测技术研究Ca2+流速的成果分析方法生物...

蚂蚁淘 /发布时间:1629221402 /浏览量:422

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

================ 蚂蚁淘在线 ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

《Science》发表非损伤微测技术研究Ca2+流速的成果 分析方法 生物...