Team:KIT-Kyoto/Protocol

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About protocol

As it is of common knowledge, iGEM is carried out within the framework of a protocol common to all teams. Whereas most of the teams are registered on this page, we have uploaded not only the English protocol but also the Japanese one, e.g., our mother tongue version. This is the common protocol with some improvements after we translated it into Japanese.Publishing the Japanese protocol in will be of help to other new Japanese teams in the future. Furthermore, this will be linked to an improvement of iGEM’s name identification in Japan and will enhance the public recognition of synthetic biology. It will also promote Science and Communication among people within not only the scientific but with the non-scientific communities as well. We believe that these improvements will constitute a contribution to the illustration of the public in general.

Contents

Japanese   English トランスフォーメーション   Bacterial transformation アルカリミニプレップ   DNA miniprep ポリメラーゼ連鎖反応(PCR)   Polymerase Chain Reaction(PCR) 制限酵素処理   DNA digestion by restriction enzymes アガロースゲル電気泳動   Agarose gel electrophoresis ゲルからのDNA抽出   Isolation of DNA fragments from agarose gel ライゲーション   DNA ligation コンピテント細胞の作製   Preparing chemically competent cells シークエンス   Sequencing グリセロールストック   Glycerol stock LB培地   LB culture medium 2×YT培地   2×YT culture medium SOB培地   SOB culture medium SOC培地   SOC culture medium Protocol トランスフォーメーション   Bacterial transformation ↓ 氷上でコンピテント細胞(DH5α:大腸菌株)を解凍する   ↓ Thaw competent cells(DH5 Alpha:E.coli strain) on ice. ↓ 前もって冷やしておいた1.5 mlチューブに100 μlのコンピテント細胞を分注する余ったコンピテント細胞は-80 °Cの冷凍庫に戻す   ↓ Aliquot 100 μl cells into pre-chilled 1.5 ml tube.Put excess competent cells back into the -80 °C freezer. ↓ DNAをチューブに1～5 μl加えて、氷上で30分間冷やす   ↓ Add DNA (1 to 5 μl) to tube, incubate on ice for 30 minutes. ↓ 42 °Cで45秒間熱ショックを与える   ↓ Heat shock the cells at 42 °C for 45 seconds. ↓ 0.9 mlのSOC培地を加える   ↓ Add 0.9 ml SOC medium. ↓ 37 °Cで1時間、振りながら回復培養する   ↓ Rescue at 37 ℃ for 1 hour, shaking. ↓ 1 mlをLBプレート(+amp,+kan,+camのいずれか)にまく   ↓ Spread 1 ml on LB plates(Either +amp,+kan or +cam) ↓ 37 °Cで一晩培養する   ↓ Clutivate overnight in 37 °C. >>Contents