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...)
Current revision (10:58, 13 August 2009) (view source)
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==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 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.
* 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.
** 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
 +
 +
==Transposome DNA construction using PCR and cutting==
 +
 +
* 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
 +
 +
==Transposome DNA construction using PCR==
 +
 +
* 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)
 +
 +
 +
==Final transposome construction==
 +
* 1 μL transposon DNA, with phosphorylated ME ends, 100 ng/μL
 +
* 2 μL EZ-Transposase (Epicentre) [http://epibio.com/item.asp?ID=292]
 +
* 1 μL glycerol
 +
* hold at RT for 1/2 hour
 +
* reported to age at 4C overnight to provide higher efficiency
 +
 +
 +
[[category:Mesoplasma florum]][[category:protocol]]

Current revision

Contents

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 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

Transposome DNA construction using PCR and cutting

  • 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

Transposome DNA construction using PCR

  • 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)


Final transposome construction

  • 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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