Mesoplasma florum:Transposome construction

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(New page: * 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...)
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** Use NEB Buffer 1 with BSA which is good for both Sau3AI and PvuII.
** Use NEB Buffer 1 with BSA which is good for both Sau3AI and PvuII.
* TT01 transposon is 2478 bp long
* 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

Revision as of 15:16, 1 October 2007

  • 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
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