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Team:HokkaidoU Japan/Notebook/October2
From 2010.igem.org
(Difference between revisions)
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<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/October1|October 1]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/October3|October 3]]</div></div> | <div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/October1|October 1]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/October3|October 3]]</div></div> | ||
| - | == Colony PCR == | + | ==Colony PCR== |
[[Image:HokkaidoU Japan 20101002a.JPG|200px|right|thumb]] | [[Image:HokkaidoU Japan 20101002a.JPG|200px|right|thumb]] | ||
* Performed Colonie PCR for 3 colonies which were incubated over night | * Performed Colonie PCR for 3 colonies which were incubated over night | ||
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** but when result came, had already done miniprep and prepared Sequencing Master Mix | ** but when result came, had already done miniprep and prepared Sequencing Master Mix | ||
<div style="clear:both;"></div> | <div style="clear:both;"></div> | ||
| - | == Miniprep == | + | ==Miniprep== |
* Used Qiagen kit | * Used Qiagen kit | ||
* Melted in 50 uL TE instead of H2O | * Melted in 50 uL TE instead of H2O | ||
| - | == Preparation for Sequencing == | + | ==Preparation for Sequencing== |
* Mixed as shown in the table below | * Mixed as shown in the table below | ||
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|} | |} | ||
| + | =3 Piece Ligationやり直し= | ||
[[Image:HokkaidoU Japan 20101002b.JPG|200px|right|thumb]] | [[Image:HokkaidoU Japan 20101002b.JPG|200px|right|thumb]] | ||
| + | ==ゲル抽== | ||
| + | 10月1日に制限酵素処理したDNA solutionをゲル抽 | ||
| + | *左からλ/''Hin''dIII, pSB1T3, pSB1A3, Arabinose Promoter([[Team:HokkaidoU_Japan/Material_And_Methods#Materials_And_Methods|3-20B]]), T3SS signal, T3SS signal | ||
| + | ** T3SS signalはバンドが見えないため，制限酵素処理からやり直し．原因不明． | ||
| + | <div style="clear:both;"></div> | ||
| + | |||
| + | =Colony PCR= | ||
[[Image:HokkaidoU Japan 20101002c.JPG|200px|right|thumb]] | [[Image:HokkaidoU Japan 20101002c.JPG|200px|right|thumb]] | ||
[[Image:HokkaidoU Japan 20101002d.JPG|200px|right|thumb]] | [[Image:HokkaidoU Japan 20101002d.JPG|200px|right|thumb]] | ||
[[Image:HokkaidoU Japan 20101002e.JPG|200px|right|thumb]] | [[Image:HokkaidoU Japan 20101002e.JPG|200px|right|thumb]] | ||
Revision as of 15:22, 2 October 2010
since 13 Jul 2010
since 1 Aug 2010
Colony PCR
- Performed Colonie PCR for 3 colonies which were incubated over night
- Colony numbers were: 1, 2 and 3
- Results showed no insertion
- but when result came, had already done miniprep and prepared Sequencing Master Mix
Miniprep
- Used Qiagen kit
- Melted in 50 uL TE instead of H2O
Preparation for Sequencing
- Mixed as shown in the table below
5x Sequencing Buffer 1.5uL 24.75 uL Ready Reaction Premix 1 uL 16.5 H2O 5 uL 80 uL total 7.5/sample
| Reagent | Amount | Amount for 16.5 |
|---|---|---|
| 5x Sequencing Buffer | 1.5uL | 24.75 uL |
| Ready Reaction Premix | 1 uL | 16.5 |
| H2O | 5 uL | 80 uL |
| Total | 7.5/sample | 121.25 |
3 Piece Ligationやり直し
ゲル抽
10月1日に制限酵素処理したDNA solutionをゲル抽
- 左からλ/HindIII, pSB1T3, pSB1A3, Arabinose Promoter(3-20B), T3SS signal, T3SS signal
- T3SS signalはバンドが見えないため，制限酵素処理からやり直し．原因不明．
Colony PCR
