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 トランスフォーメーション アルカリミニプレップ ポリメラーゼ連鎖反応(PCR) 制限酵素処理 アガロースゲル電気泳動 ゲルからのDNA抽出 ライゲーション コンピテント細胞の作製 シークエンス グリセロールストック LB培地 2xYT培地 SOB培地 SOC培地

English Bacterial transformation DNA miniprep Polymerase Chain Reaction(PCR) DNA digestion by restriction enzymes Agarose gel electrophoresis Isolation of DNA fragments from agarose gel DNA ligation Preparing chemically competent cells Sequencing Glycerol stock LB culture medium 2xYT culture medium SOB culture medium SOC culture medium

Protocol

</td> </td> </td> </td> </td> </td> </td> トランスフォーメーション ↓氷上でコンピテント細胞(DH5α:大腸菌株)を融かす ↓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 ℃の冷凍庫に戻す ↓DNAをチューブに1～5 μl加えて、氷上で30分間冷やす ↓42 ℃で45秒間熱ショックを与える ↓0.9 mlのSOC培地を加える ↓37 ℃で1時間、振りながら回復培養する ↓1 mlをLBプレート(+amp,+kan,+camのいずれか)にまく ↓37 ℃で一晩培養する

</td> </td> </td> </td> </td> </td> </td> Bacterial transformation ↓Thaw competent cells(DH5 Alpha:E.coli strain) on ice. ↓Aliquot 100 μl cells into pre-chilled 1.5 ml tube.Put excess competent cells back into the -80℃ freezer. ↓Add DNA (1 to 5 μl) to tube, incubate on ice for 30 minutes. ↓Heat shock the cells at 42 ℃ for 45 seconds. ↓Add 0.9 ml SOC medium. ↓Rescue at 37 ℃ for 1 hour, shaking. ↓Spread 1 ml on LB plates(Either +amp,+kan or +cam) ↓Clutivate overnight in 37 ℃ . >>Contents