Team:KIT-Kyoto/Protocol

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Language : English / Japanese

About protocol

PROT.PNG
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(DH5 Alpha: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 °C 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 ↓ Cultivate overnight in 37 °C.
アルカリミニプレップ  DNA miniprep
Solution I50 mM グルコース (MW 180)
 10 mM EDTA(pH 8.0)
 25 mM Tris-HCl (pH 8.0)
Solution II0.2 N NaOH
 1% SDS
Solution III3 M 酢酸カリウム
 1.8 M 酢酸
 
Solution I50 mM glucose (MW 180)
 10 mM EDTA(pH 8.0)
 25 mM Tris-HCl (pH 8.0)
Solution II0.2 N NaOH
 1% SDS
Solution III3 M potassium acetate
 1.8 M acetic acid
↓ LBプレート(+amp,+kan,+camのいずれか)に大腸菌をまき、37 °Cで一晩培養する ↓ Streak E.coli to LB plates(Either +amp,+kan or +cam) and cultivate overnight in 37 °C.
↓ プレートからシングルコロニーを分離する ↓ Pick up a single colony from plate.
↓ 2 mlのLB培地で37 °Cで一晩振とう培養する ↓ Cultivate in 2 ml LB medium overnight in 37 °C, shaking.
↓ 1.5 mlの培養液を1.5 mlチューブにうつす ↓ Pipet 1.5 ml of overnight culture into a 1.5 ml tube.
↓ 15,000 rpm、4 °Cで1分間遠心し、 上清を捨てる ↓ Centrifuge for 1 minute at 15,000 rpm in 4 °C and discard the supernatant.
↓ 100 μlの氷冷したSolution Iを沈殿に加え、懸濁する ↓ Add 100 μl of refrigerated Solution I to the pellet and vortex to resuspend.
↓ 200 μlのSolution IIを加え、混ぜる、ボルテックスは使用しない ↓ Add 200 μl of Solution II and invert gently to mix. Do not vortex.
↓ 氷上で5分間冷やす ↓ Incubate on ice for 5 minutes.
↓ 150 μlの氷冷したSolution IIIを加え、穏やかに反転し混ぜる、ボルテックスは使用しない ↓ Add 150 μl of refrigerated Solution III and invert gently to mix. Do not vortex.
↓ 氷上で5分間冷やす ↓ Incubate on ice for 5 minutes.
↓ 15,000 rpm、4 °Cで5分間遠心する ↓ Centrifuge for 5 minutes at 15,000 rpm in 4 °C.
↓ 400 μlのきれいな上清を注意してピペットで新しいチューブにとる ↓ Carefully pipet 400 μl of the clean supernatant into a new tube.
↓ 900 μlの2-プロパノールを加え、混ぜる ↓ Add 900 μl of 2-propanol and mix.
↓ 2分間室温で放置する ↓ Incubate for 2 minutes in room temperature.
↓ 15,000 rpm、4 °Cで10分間遠心し、上清を捨てる ↓ Centrifuge for 10 minutes at 15,000 rpm in 4 °C and discard supernatant.
↓ 1 mlの70%エタノールを加える ↓ Add 1 ml 70% EtOH to the pellet.
↓ 15,000 rpm、4 °Cで2分間遠心し、上清を捨てる ↓ Centrifuge for 2 minutes at 15,000 rpm in 4 °C and discard supernatant.
↓ 沈殿を10分～15分乾かす ↓ Dry the pellet for 10-15 minutes.
↓ プラスミドDNAを30 μlのRNaseのはいったTEに溶かす ↓ Resuspend the plasmid DNA in 30 μl of TE with RNase.
ポリメラーゼ連鎖反応(PCR)  Polymerase Chain Reaction(PCR)
↓ ソフトウェアプログラムを使って、プライマーの設計をする ↓ Design primers with software programs.
[http://ginkgobioworks.com/cgi/primer.cgi>>Ginkgo BioWorks] [http://ginkgobioworks.com/cgi/primer.cgi>>Ginkgo BioWorks]
↓ 下記の組成に従って試薬を混ぜる ↓ Mix the reagent according to the following components.
Primer Mix1.5 μl
鋳型 DNA0.5 μl
10 x PCR Buffer for KOD-Plus-5 μl
2 mM dNTPs5 μl
2 mM MgSO44 μl
ddH2O33 μl
KOD-Plus-1 μl
 全量 50 µl
 
Primer Mix1.5 μl
Template DNA0.5 μl
10 x PCR Buffer for KOD-Plus-5 μl
2 mM dNTPs5 μl
2 mM MgSO44 μl
ddH2O33 μl
KOD-Plus-1 μl
 50 μl system
↓ PCRのプログラム設定 ↓ PCR program
Start94 °C、2分
Cycle x 3594 °C、15秒 (熱変性)
 (Tm-10) °C、0.5分 (アニーリング)
 68 °C、1 kb/min (伸長)
End68 °C、2分
 4 °Cで保存
 
Start94 °C for 2 minutes.
Cycle x 3594 °C for 15 seconds. (denaturing)
 (Tm-10) °C for 0.5 minutes. (annealing)
 68 °C for 1 kb/min. (extension)
End68 °C for 2 minutes.
 Keep at 4 °C forever.
制限酵素処理  DNA digestion by restriction enzymes
↓ 下記の組成に従って試薬を混ぜる ↓ Mix the reagent according to the following components.
10 x Buffer2 μl
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=H-BUFF&us_id=&us_pwd=&bun_id=020010020&iv_trademark=BUF-1&kskh_kbn=01&ksk_jkn=BUF-1 H Buffer]
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=M-BUFF&us_id=&us_pwd=&bun_id=020010020&iv_trademark=BUF-1 M Buffer]
DNA溶液DNA量が1 μgになるように調整する
制限酵素3ユニットになるように調整する
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=ECO-111&us_id=&us_pwd=&bun_id=0200100100410&iv_trademark=ECO-1&kskh_kbn=01&ksk_jkn=ECO-1 EcoR I]
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=XBA-101W&us_id=&us_pwd=&bun_id=0200100100970&iv_trademark=XBA-1&kskh_kbn=01&ksk_jkn=XBA-1 Xba I]
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=PST-111&us_id=&us_pwd=?us_id=&bun_id=0200100100760&iv_trademark=PST-1&kskh_kbn=01&ksk_jkn=PST-1 Pst I]
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=SPE-101&us_id=&us_pwd=?us_id=&bun_id=0200100100900&iv_trademark=SPE-1&kskh_kbn=01&ksk_jkn=SPE-1 Spe I]
ddH2O全量が20 μlになるように調整する
 全量 20 μl
 
10 x Buffer2 µl
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=H-BUFF&us_id=&us_pwd=&bun_id=020010020&iv_trademark=BUF-1&kskh_kbn=01&ksk_jkn=BUF-1 H Buffer]
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=M-BUFF&us_id=&us_pwd=&bun_id=020010020&iv_trademark=BUF-1 M Buffer]
DNA solutionAdjust for the amount of DNA to become 1 μg.
Restriction enzymeAdjust to become 3 units.
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=ECO-111&us_id=&us_pwd=&bun_id=0200100100410&iv_trademark=ECO-1&kskh_kbn=01&ksk_jkn=ECO-1 EcoR I]
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=XBA-101W&us_id=&us_pwd=&bun_id=0200100100970&iv_trademark=XBA-1&kskh_kbn=01&ksk_jkn=XBA-1Xba I]
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=PST-111&us_id=&us_pwd=?us_id=&bun_id=0200100100760&iv_trademark=PST-1&kskh_kbn=01&ksk_jkn=PST-1 Pst I]
[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=SPE-101&us_id=&us_pwd=?us_id=&bun_id=0200100100900&iv_trademark=SPE-1&kskh_kbn=01&ksk_jkn=SPE-1 Spe I]
ddH2OAdjust to become total 20 μl.
 20 μl system
↓ 37 °Cで1時間放置する ↓ Incubate for 1 hour in 37 °C.
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