Team:HokkaidoU Japan/Notebook/August19

From 2010.igem.org

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前日のLigationでコロニーができなかったので，ベクターに問題があるかもしれない．<br>
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* pSB1C3 2 uLにligation solution 2 uLとT4 ligase 0.4 uLを加えた
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Because there weren`t any colonies the previous day we suspected that there might be something wrong with vectors<br>
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* Transformation
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* Mixed 2 uL of pSB1C3, 2 uL of ligation solution and 0.4 uL of T4 ligase
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* Transformed

Revision as of 17:36, 21 September 2010

Notebook

Ligation

VectorにHeat shock promotorとRBSを組み込む

パーツ By comparison to marker size (bp) (ng) required (uL) used
Vector 5 ng/uL 2996 bp 10 ng 2 uL
Heat shock promotor 25 ng/uL 800 bp 8 ng 0.3 uL
RBS 1 ng/uL 50 bp 1 ng 1 uL
Total 3.3 uL

Ligation and transformation follows same protocol as previously

Competency Check

  1. Transfered 1 uL of pUC119 DNA (0.35 ug/uL) into 0.5 mL tube and added 2.5 uL of ADW (0.1 ug/uL)
  2. Took new 1.5 mL tube and added 999 uL of ADW (0.1 ng/uL) to it plus 1 uL of [1]
  3. Took new 1.5 mL tube and added 99 uL of ADW to it plus 1 uL of [2]
  • Added pUC119 DNA (1 pg/uL) to each DH5alpha tube of 50ul and 30ul also varying the amount of pUC119 DNA:1 uL, 10 uL, 100 uL
  • Transformed

Ligation and Transformation between Vector，Transformation

Because there weren`t any colonies the previous day we suspected that there might be something wrong with vectors

  • Mixed 2 uL of pSB1C3, 2 uL of ligation solution and 0.4 uL of T4 ligase
  • Transformed
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