Team:KIT-Kyoto/Protocol

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Latest revision as of 15:33, 26 October 2010

Home > Notebook > Protocol

Language : English / Japanese

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.

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		↓ Recover 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.

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アルカリミニプレップ

DNA miniprep

Solution I	50 mM グルコース (MW 180)
	10 mM EDTA(pH 8.0)
	25 mM Tris-HCl (pH 8.0)
Solution II	0.2 N NaOH
	1% SDS
Solution III	3 M 酢酸カリウム
	1.8 M 酢酸

Solution I	50 mM glucose (MW 180)
	10 mM EDTA(pH 8.0)
	25 mM Tris-HCl (pH 8.0)
Solution II	0.2 N NaOH
	1% SDS
Solution III	3 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.

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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]

↓ 下記の組成に従って試薬を混ぜる

↓ Mix the reagent according to the following components.

Primer Mix	1.5 μl
鋳型 DNA	0.5 μl
10 x PCR Buffer for KOD-Plus-	5 μl
2 mM dNTPs	5 μl
2 mM MgSO₄	4 μl
ddH₂O	33 μl
KOD-Plus-	1 μl
	全量 50 µl

Primer Mix	1.5 μl
Template DNA	0.5 μl
10 x PCR Buffer for KOD-Plus-	5 μl
2 mM dNTPs	5 μl
2 mM MgSO₄	4 μl
ddH₂O	33 μl
KOD-Plus-	1 μl
	50 μl system

↓ PCRのプログラム設定

↓ PCR program

Start	94 °C、2分
Cycle x 35	94 °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)
End	68 °C、2分
End	4 °Cで保存

Start	94 °C for 2 minutes.
Cycle x 35	94 °C for 15 seconds. (denaturing)
	(Tm-10) °C for 0.5 minutes. (annealing)
	68 °C for 1 kb/min. (extension)
End	68 °C for 2 minutes.
End	Keep at 4 °C forever.

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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 Buffer	2 μl (全量の10分の1)
[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-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]
ddH₂O	全量が20 μl になるように調整する
	全量 20 μl

10 x Buffer	2 µl (1/10 of total)
[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 solution	Adjust for the amount of DNA to become 1 μg.
Restriction enzyme	Adjust 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]
ddH₂O	Adjust 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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アガロースゲル電気泳動

Agarose gel electrophoresis

50 x TAE	Tris base 242 g
	無水酢酸 57.1 ml
	0.5 M EDTA (pH 8.0) 100ml
	ddH₂Oを加えて1 Lにする

50 x TAE	242g Tris base
	57.1 ml of glacial Acetic acid
	100ml of 0.5 M EDTA (pH 8.0)
	Make up to 1 L with ddH₂O

1 x TAEを作るには、20 mlの50 x TAEに980 mlのH₂Oを加える

To make 1 x TAE from 50 x TAE stock, dilute 20 ml of stock into 980 ml of ddH₂O.

↓ ゲルに使用するアガロースの量は扱うDNAによって変わるので下記の表に従えばよい

↓ The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide.

アガロース濃度 (g/100 ml)	最適なDNAの長さ(kb)
0.5	1 - 30
0.7	0.8 - 12
1.0	0.5 - 10
1.2	0.4 - 7
1.5	0.2 - 3

Agarose Concentration (g/100 ml)	Optimal DNA Length (kb)
0.5	1 - 30
0.7	0.8 - 12
1.0	0.5 - 10
1.2	0.4 - 7
1.5	0.2 - 3

例えば、1%ゲルを作るには、1 gのアガロースをフラスコに量りとり、100 mlの1 x TAEを加える

For example,to make a 1%agarose gel,weigh out 1 g of agarose into a flask and add 100 ml of 1 x TAE.

↓ 電子レンジでアガロースが完全に溶けるまで加熱する

↓ Heat solution in a microwave until agarose is completely dissolved.

↓ 素手でフラスコの底が触れるまで冷ます

↓ Let the agarose cool until touching the bottom of a flask with your bare hand.

↓ ゲルトレーにゲルを注ぎ、コームを挿す

↓ Pour into gel tray and insert comb(s).

↓ 室温で15分から30分間冷ます

↓ Allow to cool for 15-30 minutes at room temperature.

↓ コームを外し、電気泳動槽にうつして、1 x TAEに浸す

↓ Remove comb(s),place in electrophoresis chamber and cover with 1 x TAE.

↓ サンプルに6 x ローディングダイを加える

↓ Add 6 x loading dye to samples.

↓ DNAとマーカー(100 bp DNA ladder,1 kb DNA ladder)をゲルにのせる

↓ Load DNA and standard (100 bp DNA ladder,1 kb DNA ladder) onto gel.

↓ 100 Vで20分間電気泳動する

↓ Electrophorese at 100 V for 20 minutes.

↓ EtBrでゲルを30分間染色する

↓ Dye gel with EtBr for 30 minutes.

↓ UVを照射してDNAのバンドを可視化する

↓ Visualize DNA bands using UV lightbox.

>>back to Contents

ゲルからのDNA抽出		Isolation of DNA fragments from agarose gel
[http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx QIAquick Gel Extraction Kit]を使用する		We use [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx QIAquick Gel Extraction Kit].
↓ 1 x TAEに溶かした低融点のアガロースゲルをつくる		↓ Make a low-melt agarose gel in 1 x TAE.
↓ DNAとマーカーをゲルにのせる		↓ Load DNA and standard onto gel.
↓ 75 Vで45分間電気泳動する		↓ Electrophorese at 75 V for 45 minutes.
↓ EtBrでゲルを40分間染色する		↓Dye gel with EtBr for 40 minutes.
↓ UVを照射してDNAのバンドを可視化する		↓ Visualize DNA bands using UV lightbox.
↓ 清潔で鋭利な手術用メスでゲルからDNA断片を切りだす		↓ Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
↓ 1.5 mlチューブ中のゲルスライスの重さを量る		↓ Weigh the gel slice in 1.5 ml tube.
↓ ゲル100 mgに対して、3倍量(300 μl)のBuffer QGを加える		↓ Add 3 volumes of Buffer QG to 1 volume of gel (100 mg - 100 μl).
↓ 42-50 °Cで10分間放置し、ゲルを完全に溶かす		↓ Incubate at 42-50 °C for 10 minutes or until gel is fully dissolved.
↓ ゲルが完全に溶けたら、溶液が黄色であることを確認する		↓ After the gel slice has dissolved completely, check that the color of the mixture is yellow.
↓ ゲル100 mgに対して、当量(100 μl)の2-プロパノールを加え、混ぜる		↓ Add 1 gel volume of 2-propanol to the sample and mix.
↓ カラムにサンプルをのせる		↓ Apply sample to column in a collection tube.
↓ 15,000 rpmで1分間遠心し、ろ液を捨てる		↓ Centrifuge for 1 minute at 15,000 rpm and discard flow-through.
↓ 500 μlのBuffer QGをカラムに加え、残りのゲルを溶かす		↓ Add 500 μl of Buffer QG to column to dissolve residual agarose.
↓ 15,000 rpmで1分間遠心し、ろ液を捨てる		↓ Centrifuge for 1 minute at 15,000 rpm and discard flow-through.
↓ 750 μlのwash Buffer PEをカラムに加える		↓ Add 750 μl of wash Buffer PE to column.
↓ 15,000 rpmで1分間遠心し、ろ液を捨てる		↓Centrifuge for 1 minute at 15,000 rpm and discard flow-through.
↓ 15,000 rpmで1分間遠心し、ろ液を捨てる(残りのエタノールを除くため)		↓ Centrifuge for 1 minute at 15,000 rpm and discard flow-through (to eliminate residual ethanol).
↓ カラムを新しい1.5 mlチューブに移しかえる		↓ Place column into a clean 1.5 ml tube.
↓ 30 μlのelution Buffer EBをカラムの中央に加える		↓ Add 30 μl of elution Buffer EB to center of column.
↓ 1分間立たせておく		↓Let stand for 1 minute.
↓ 15,000 rpmで1分間遠心する		↓ Centrifuge for 1 minute at 15,000 rpm.
↓ 集めたDNAは1.5 mlチューブに入れて-20 °Cで保存する		↓ Store DNA collected in 1.5 ml tube at -20 °C.

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ライゲーション

DNA ligation

Rapid DNA Ligation Kitを使用する

We use Rapid DNA Ligation Kit.

↓ 下記の組成に従って試薬を混ぜる

↓ Mix the reagent according to the following components.

5 x DNA dilution buffer	2 μl
ベクター(カット・CHIP処理後)	0.25-1 μl
インサート DNA	0.5-6.5 μl
ddH₂O	全量が10 μlになるように調整する

5x DNA dilution buffer	2 μl
Vector(cut and CHIP treated)	0.25-1 μl
Insert DNA	0.5-6.5 μl
ddH₂O	To final volume of 10 μl

↓ よく混ぜる

↓ Mix well.

↓ 10 μlの2 x rapid ligation bufferと1 μlのligaseを加える

↓ Add 10 μl of 2 x rapid ligation buffer and 1 μl ligase.

↓ よく混ぜる

↓ Mix well.

↓ 室温で5分間放置する

↓ Hold at room temperature for 5 minutes.

↓ トランスフォーメーションに使用するときは、100 μlのコンピテント細胞に対して10 μl使用する

↓Then use 10 μl of the ligation reaction to transform 100 μl of chemically competent cells.

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コンピテント細胞の作製		Preparing chemically competent cells
↓ E.coli(DH5 Alpha)をLBプレートにまき、37 °Cで一晩培養する		↓ Streak E.coli (DH5 Alpha) to LB plate and cultivate overnight in 37 °C.
↓ シングルコロニーをピックアップして、2 mlのSOB培地で37 °Cで6時間培養する		↓ Pick up a single colony from plate and cultivate 2 ml of SOB medium for 6 hours in 37 °C.
↓ 培養液を250 mlのSOB培地に加える		↓ Add 2 ml of the culture 250 ml of SOB medium.
↓ 遠心しながら、37 °Cで吸光度(OD600)が0.4-0.8になるまで培養する		↓ Cultivate at 37°C with agitation until the Optical Density (OD600) reaches 0.4-0.8.
↓ 培養液を50 mlの遠心チューブに移し、氷上で10分間冷やす		↓ Transfer the culture into 50 ml centrifuge tube and incubate the culture on ice for 10 minutes.
↓ 4,500 rpm、4 °Cで10分間遠心し、上清を捨てる		↓ Centrifuge for 10 minutes at 4,500 rpm in 4 °C and discard the supernatant.
↓ 84 mlの氷冷した[http://openwetware.org/wiki/TB_buffer TB Buffer]を加えて、懸濁する		↓ Add 84 ml of refrigerated [http://openwetware.org/wiki/TB_buffer TB Buffer] and resuspend.
↓ 氷上で10分間冷やす		↓ Incubate on ice for 10 minutes.
↓ 4,500 rpm、4 °Cで10分間遠心し、上清を捨てる		↓ Centrifuge for 10 minutes at 4,500 rpm in 4 °C and discard the supernatant.
↓ 20 mlの氷冷した[http://openwetware.org/wiki/TB_buffer TB Buffer]を加えて、懸濁する		↓ Add 20 ml of refrigerated [http://openwetware.org/wiki/TB_buffer TB Buffer] and resuspend.
↓ 1.4 mlの7% DMSO(1.4 mlのDMSOに18.6 mlのTB Bufferを加えたもの)を加える		↓ Add 1.4 ml of 7% DMSO(1.4 ml of DMSO to 18.6 ml of TB).
↓ 氷上で10分間冷やす		↓ Incubate on ice for 10 minutes.
↓ 300 μlずつ分注する		↓ Aliquot 300 μl of the cells.
↓ 液体窒素で冷凍する		↓ Freeze the cells in liquid nitrogen.
↓ -80 °Cで保存する		↓ Store at -80 °C.

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シークエンス

Sequencing

PCR

↓ 下記の組成に従って試薬を混ぜる

↓ Mix the reagent according to the following components.

H₂O	全量10 μlになるように調整する
Premix	2 μl
5 x sequence buffer	1 μl
Primer(1 μM)	1.6 μl
プラスミド DNA	DNA量が100-250 ngになるように調整する
	全量 10 μl

H₂O	Adjust to become total 10 μl
Premix	2 μl
5 x sequence buffer	1 μl
Primer(1 μM)	1.6 μl
Plasmid DNA	Adjust for the amount of DNA to become about 100-250 ng.
	10 μl system

↓ PCRのプログラム設定

↓ PCR program

Start	96 °C、1分
Cycle x 30	96 °C、10秒
	50 °C、5秒
	60 °C、4分
End	4 °Cで保つ

Start	96 °C for 1 minute.
Cycle x 30	96 °C for 10 seconds.
	50 °C for 5 seconds.
	60 °C for 4 minutes.
End	Keep at 4 °C forever.

エタノール沈殿

EtOH precipitation

↓ PCRの後、1.5 mlチューブに移す

↓ Remove 1.5 ml tube after PCR.

↓ 1.5 μlの3 M 酢酸ナトリウムを加える

↓ Add 1.5 μl of 3 M CH3COONa.

↓ 氷上で15分間冷やす

↓ Incubate on ice for 15 minutes.

↓ 31.5 μlの2-プロパノールと7 μlのH₂Oを加える

↓ Add 31.5 μl of 2-propanol and 7 μl of H₂O.

↓ 15,000 rpm、4 °Cで20分間遠心し、上清を捨てる

↓ Centrifuge for 20 minutes at 15,000 rpm in 4 °C and discard the supernatant.

↓ 50 μlの70% エタノールを加える.

↓ Add 50 μl of 70% EtOH.

↓ 15,000 rpm、4 °Cで20分間遠心し、上清を捨てる

↓ Centrifuge for 20 minutes at 15,000 rpm in 4 °C and discard the supernatant.

↓ 乾燥させる(よく乾かす)

↓ Dry up.(Dry it well.)

↓ 15 μlのHi-Di formamideに溶かす

↓ Dissolve in 15 μl of Hi-Di formamide.

↓ 95 °Cで2分加熱する

↓ Incubate for 2 minutes at 95 °C.

↓ 氷上で冷やす(急冷)

↓ Incubate on ice(rapid cooling).

↓ 0.5 mlチューブの蓋を切り取る

↓ Cut out the cap of the 0.5 ml tube.

↓ サンプルを1.5 mlチューブから0.5 mlチューブに移す

↓ Remove the sample in the 1.5 ml tube to 0.5 ml tube.

↓ シークエンス用の蓋をする

↓ Do the cap for the sequencing.

↓ シークエンサーに入れる

↓ Take the sample in to sequencer.

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グリセロールストック		Glycerol stock
↓ E.coli (DH5 Alpha)をLBプレートにまき、37 °Cで一晩培養する		↓ Streak E.coli (DH5 Alpha) to LB plate and cultivate overnight in 37 °C.
↓ シングルコロニーをピックアップして、2 mlのLB培地で37 °Cで一晩培養する		↓ Pick up a single colony from plate and cultivate 2 ml of LB medium overnight in 37 °C.
↓ 1.5 mlチューブ内で500 μlの培養液を500 μLの50%グリセロールに加える		↓ Add 500 μl of an overnight culture to 500 μl of 50% glycerol in a 2 mL screw top tube.
↓ -80 °Cでグリセロールストックを冷凍する		↓ Freeze the glycerol stock tube at -80 °C.

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LB培地

LB culture medium

↓ 下記の組成に従って試薬を混ぜる

↓Mix the reagent according to the following components.

Bacto Tryptone	10 g
Extract of Yeast	5 g
NaCl	1 g
H₂O	1 L

Bacto Tryptone	10 g
Extract of Yeast	5 g
NaCl	1 g
H₂O	1 L

↓ 121 °Cで20分間オートクレーブする

↓ Autoclave for 20 minutes at 121 °C.

LBプレートを作るときは、20 gの寒天を加え、オートクレーブ後、20 mlずつプレートに温かい培地を注ぐ

If you make LB plates, add 20 g of agar and after autoclave pour 20 ml of warm media into each plate.

抗生物質入りのプレートを作るときは、オートクレーブ後、冷めた培地に抗生物質を加える

If you make LB plates with the antibiotic, after autoclave add the antibiotic to the medium cooled.

>>back to Contents

2 x YT培地

2 x YT culture medium

↓ 下記の組成に従って試薬を混ぜる

↓ Mix the reagent according to the following components.

Bacto Tryptone	16 g
Extract of Yeast	10 g
NaCl	5 g
H₂O	1 L

Bacto Tryptone	16 g
Extract of Yeast	10 g
NaCl	5 g
H₂O	1 L

↓ 121 °Cで20分間オートクレーブする

↓ Autoclave for 20 minutes at 121 °C.

>>back to Contents

SOB培地

SOB culture medium

↓ 下記の組成に従って試薬を混ぜる

↓ Mix the reagent according to the following components.

Bacto Tryptone	20 g
Extract of Yeast	5g
5M NaCl	2ml
2M KCl	1.25 ml
H₂O	1 L

Bacto Tryptone	20 g
Extract of Yeast	5g
5M NaCl	2ml
2M KCl	1.25 ml
H₂O	1 L

↓ 121 °Cで20分間オートクレーブする

↓ Autoclave for 20 minutes at 121 °C.

↓ 10 mlの2M Mg solution (1 M MgSO₄・7H₂O +1 M MgCl₂・6H₂O)を加える

↓ Add 10 ml of 2M Mg solution (1 M MgSO₄・7H₂O +1 M MgCl₂・6H₂O).

>>back to Contents

SOC培地

SOC culture medium

↓ 下記の組成に従って試薬を混ぜる

↓ Mix the reagent according to the following components.

Bacto Tryptone	20 g
Extract of Yeast	5g
5M NaCl	2ml
2M KCl	1.25 ml
H₂O	1 L

Bacto Tryptone	20 g
Extract of Yeast	5g
5M NaCl	2ml
2M KCl	1.25 ml
H₂O	1 L

↓ 121 °Cで20分間オートクレーブする

↓ Autoclave for 20 minutes at 121 °C.

↓ 10 mlの2M Mg solution (1 M MgSO₄・7H₂O +1 M MgCl₂・6H₂O)を加える

↓ Add 10 ml of 2M Mg solution (1 M MgSO₄・7H₂O +1 M MgCl₂・6H₂O).

↓ 培地が50 °C以下に冷めた後、20 mlの滅菌済みの20%グルコース溶液を加える

↓ After cooling medium to less than 50°C, add 20 ml filter sterilized 20% glucose solution.

>>back to Contents

@@ Line 1: / Line 1: @@
 {{Template:KIT-Kyoto/menu}}
 <table border=0 width="965px" align="center"><tr><td>
-<div aling="left">[[Team:KIT-Kyoto|Home]] > [[Team:KIT-Kyoto/Note|Notebook]] > [[Team:KIT-Kyoto/Protocol|Protocol]]</div></td><td><div align="right">Language : [[Team:KIT-Kyoto/Protocol|English]] / [[Team:KIT-Kyoto/ProtocolJ|Japanese]]</div></td></tr></table>
+<div aling="left">[[Team:KIT-Kyoto/Home|Home]] > [[Team:KIT-Kyoto/Note|Notebook]] > [[Team:KIT-Kyoto/Protocol|Protocol]]</div></td><td><div align="right">Language : [[Team:KIT-Kyoto/Protocol|English]] / [[Team:KIT-Kyoto/ProtocolJ|Japanese]]</div></td></tr></table>
 <table border="0" width="965px" align="center"><tr><td width="165px" valign="top"
@@ Line 11: / Line 11: @@
 <table width="700px" align=center>
 <tr><td>
-[[Image:PROT.PNG|left]]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.
+[[Image:PROT.PNG|left]]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.
 </td></tr></table>
@@ Line 53: / Line 53: @@
 <tr><td>↓ 42 °Cで45秒間熱ショックを与える</td><td>&nbsp;</td><td>↓ Heat shock the cells at 42 °C for 45 seconds. </td></tr>
 <tr><td>↓ 0.9 mlのSOC培地を加える</td><td>&nbsp;</td><td>↓ Add 0.9 ml SOC medium.</td></tr>
-<tr><td>↓ 37 °Cで1時間、振りながら回復培養する</td><td>&nbsp;</td><td>↓ Rescue at 37 °C for 1 hour, shaking.</td></tr>
+<tr><td>↓ 37 °Cで1時間、振りながら回復培養する</td><td>&nbsp;</td><td>↓ Recover at 37 °C for 1 hour, shaking.</td></tr>
 <tr><td>↓ 1 mlをLBプレート(+amp,+kan,+camのいずれか)にまく</td><td>&nbsp;</td><td>↓ Spread 1 ml on LB plates(Either +amp,+kan or +cam)</td></tr>
 <tr><td>↓ 37 °Cで一晩培養する</td><td>&nbsp;</td><td>↓ Cultivate overnight in 37 °C.</td></tr></table>
@@ Line 70: / Line 70: @@
 <span id="al">'''アルカリミニプレップ'''</span></td><td width="20px">&nbsp;</td><td width="340px">
 <span id="aleng">'''DNA miniprep'''</span></td></tr>
-<tr><td><table border=1 width="300px"><tr><td width="100px" align=center>Solution I</td><td width="200px"  align=right>50 mM グルコース (MW 180)</td></tr><tr><td>&nbsp;</td><td align=right>10 mM EDTA(pH 8.0)</td></tr><tr><td>&nbsp;</td><td align=right>25 mM Tris-HCl (pH 8.0)</td></tr>
+<tr><td><table border=1 width="300px"><tr><td width="100px" align=center rowspan=3>Solution I</td><td width="200px"  align=right>50 mM グルコース (MW 180)</td></tr><tr><td align=right>10 mM EDTA(pH 8.0)</td></tr><tr><td align=right>25 mM Tris-HCl (pH 8.0)</td></tr>
-<tr><td align=center>Solution II</td><td align=right>0.2 N NaOH</td></tr><tr><td>&nbsp;</td><td align=right>1% SDS</td></tr><tr><td align=center>Solution III</td><td align=right>3 M 酢酸カリウム</td></tr><tr><td>&nbsp;</td><td align=right>1.8 M 酢酸</td></tr></table></td><td>&nbsp;</td><td><table
+<tr><td align=center rowspan=2>Solution II</td><td align=right>0.2 N NaOH</td></tr><tr><td align=right>1% SDS</td></tr>
-border=1 width="300px"><tr><td width="100px" align=center>Solution I</td><td width="200px" align=right>50 mM glucose (MW 180)</td></tr>
+<tr><td align=center rowspan=2>Solution III</td><td align=right>3 M 酢酸カリウム</td></tr><tr><td align=right>1.8 M 酢酸</td></tr></table></td><td>&nbsp;</td><td><table
-<tr><td>&nbsp;</td><td align=right>10 mM EDTA(pH 8.0)</td></tr><tr><td>&nbsp;</td><td align=right>25 mM Tris-HCl (pH 8.0)</td></tr>
+border=1 width="300px"><tr><td width="100px" align=center rowspan=3>Solution I</td><td width="200px" align=right>50 mM glucose (MW 180)</td></tr>
-<tr><td align=center>Solution II</td><td align=right>0.2 N NaOH</td></tr><tr><td>&nbsp;</td><td align=right>1% SDS</td></tr><tr><td align=center>Solution III</td><td align=right>3 M potassium acetate</td></tr><tr><td>&nbsp;</td><td align=right>1.8 M acetic acid</td></tr></table></td></tr>
+<tr><td align=right>10 mM EDTA(pH 8.0)</td></tr><tr><td align=right>25 mM Tris-HCl (pH 8.0)</td></tr>
+<tr><td align=center rowspan=2>Solution II</td><td align=right>0.2 N NaOH</td></tr><tr><td align=right>1% SDS</td></tr><tr><td align=center rowspan=2>Solution III</td><td align=right>3 M potassium acetate</td></tr><tr><td align=right>1.8 M acetic acid</td></tr></table></td></tr>
 <tr><td>↓ 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</td><td>&nbsp;</td><td>↓ Streak <I>E.coli</I> to LB plates(Either +amp,+kan or +cam) and cultivate overnight in 37 °C.</td></tr>
 <tr><td>↓ プレートからシングルコロニーを分離する</td><td>&nbsp;</td><td>↓ Pick up a single colony from plate.</td></tr>
@@ Line 112: / Line 113: @@
 <tr><td><table border=1 width="300px"><tr><td width="200px" align=center>Primer Mix</td><td width="100px" align=right>1.5 μl</td></tr>
 <tr><td align=center>鋳型 DNA</td><td align=right>0.5 μl</td></tr><tr><td align=center>10 x PCR Buffer for KOD-Plus-</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM dNTPs</td><td align=right>5 μl</td></tr>
-<tr><td align=center>2 mM MgSO4</td><td align=right>4 μl</td></tr><tr><td align=center>ddH2O</td><td align=right>33 μl</td></tr><tr><td align=center>KOD-Plus-</td><td align=right>1 μl</td></tr>
+<tr><td align=center>2 mM MgSO<sub>4</sub></td><td align=right>4 μl</td></tr><tr><td align=center>ddH<sub>2</sub>O</td><td align=right>33 μl</td></tr><tr><td align=center>KOD-Plus-</td><td align=right>1 μl</td></tr>
-<tr><td>&nbsp;</td><td align=right>全量 50 µl</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td width="200px" align=center>Primer Mix</td><td width="100px" align=right>1.5 μl</td></tr><tr><td align=center>Template DNA</td><td align=right>0.5 μl</td></tr><tr><td align=center>10 x PCR Buffer for KOD-Plus-</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM dNTPs</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM MgSO4</td><td align=right>4 μl</td></tr><tr><td align=center>ddH2O</td><td align=right>33 μl</td></tr><tr><td align=center>KOD-Plus-</td><td align=right>1 μl</td></tr><tr><td>&nbsp;</td><td align=right>50 μl system</td></tr></table></td></tr>
+<tr><td>&nbsp;</td><td align=right>全量 50 µl</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td width="200px" align=center>Primer Mix</td><td width="100px" align=right>1.5 μl</td></tr><tr><td align=center>Template DNA</td><td align=right>0.5 μl</td></tr><tr><td align=center>10 x PCR Buffer for KOD-Plus-</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM dNTPs</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM MgSO<sub>4</sub></td><td align=right>4 μl</td></tr><tr><td align=center>ddH<sub>2</sub>O</td><td align=right>33 μl</td></tr><tr><td align=center>KOD-Plus-</td><td align=right>1 μl</td></tr><tr><td>&nbsp;</td><td align=right>50 μl system</td></tr></table></td></tr>
 <tr><td>↓ PCRのプログラム設定</td><td>&nbsp;</td><td>↓ PCR program</td></tr>
 <tr><td><table border=1 width="300px"><tr><td width="60px" align=center>Start</td><td width="240px" align=right>94 °C、2分</td></tr><tr><td align=center rowspan=3>Cycle x 35</td><td align=right>94 °C、15秒 (熱変性)</td></tr><tr><td align=right>(Tm-10) °C、0.5分 (アニーリング)</td></tr><tr><td align=right>68 °C、1 kb/min (伸長)</td></tr><tr><td align=center rowspan=2>End</td><td align=right>68 °C、2分</td></tr>
@@ Line 146: / Line 147: @@
 <tr><td align=center>
 :[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 <I>Spe</I> I]</td></tr>
-<tr><td align=center>ddH2O</td><td>全量が20 μl になるように調整する</td></tr>
+<tr><td align=center>ddH<sub>2</sub>O</td><td>全量が20 μl になるように調整する</td></tr>
 <tr><td>&nbsp;</td><td align=right>全量 20 μl</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px">
 <tr><td width="120px" align=center>10 x Buffer</td><td rowspan=3 width="180px">2 µl (1/10 of total)</td></tr>
@@ Line 163: / Line 164: @@
 <tr><td align=center>
 :[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 <I>Spe</I> I]</td></tr>
-<tr><td align=center>ddH2O</td><td>Adjust to become total 20 μl.</td></tr>
+<tr><td align=center>ddH<sub>2</sub>O</td><td>Adjust to become total 20 μl.</td></tr>
 <tr><td>&nbsp;</td><td align=right>20 μl system</td></tr></table></td></tr>
 <tr><td>↓ 37 °Cで1時間放置する</td><td>&nbsp;</td><td>↓ Incubate for 1 hour in 37 °C.</td></tr>
@@ Line 184: / Line 185: @@
 <span id="agareng">'''Agarose gel  electrophoresis'''</span></td></tr>
 <tr><td><table border=1 width="300px"><tr><td align=center rowspan=4 width="100px">50 x TAE</td><td width="200px">Tris base 242 g</td></tr><tr><td>無水酢酸 57.1 ml</td></tr>
-<tr><td>0.5 M EDTA (pH 8.0) 100ml</td></tr><tr><td>ddH2Oを加えて1 Lにする</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td align=center rowspan=4 width="100px">50 x TAE</td><td width="200px">242g Tris base</td></tr><tr><td>57.1 ml of glacial Acetic acid</td></tr>
+<tr><td>0.5 M EDTA (pH 8.0) 100ml</td></tr><tr><td>ddH<sub>2</sub>Oを加えて1 Lにする</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td align=center rowspan=4 width="100px">50 x TAE</td><td width="200px">242g Tris base</td></tr><tr><td>57.1 ml of glacial Acetic acid</td></tr>
-<tr><td>100ml of 0.5 M EDTA (pH 8.0)</td></tr><tr><td>Make up to 1 L with ddH2O</td></tr></table></td></tr>
+<tr><td>100ml of 0.5 M EDTA (pH 8.0)</td></tr><tr><td>Make up to 1 L with ddH<sub>2</sub>O</td></tr></table></td></tr>
-<tr><td>1 x TAEを作るには、20 mlの50 x TAEに980 mlのH2Oを加える</td><td>&nbsp;</td><td>To make 1 x TAE from 50 x TAE stock, dilute 20 ml of stock into 980 ml of ddH2O.</td></tr>
+<tr><td>1 x TAEを作るには、20 mlの50 x TAEに980 mlのH<sub>2</sub>Oを加える</td><td>&nbsp;</td><td>To make 1 x TAE from 50 x TAE stock, dilute 20 ml of stock into 980 ml of ddH<sub>2</sub>O.</td></tr>
 <tr><td>↓ ゲルに使用するアガロースの量は扱うDNAによって変わるので下記の表に従えばよい</td><td>&nbsp;</td><td>↓ The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide.</td></tr>
 <tr><td><table border=1 width="300px"><tr><td width="150px" align=center>アガロース濃度
@@ Line 241: / Line 242: @@
 <tr><td>↓ カラムにサンプルをのせる</td><td>&nbsp;</td><td>↓ Apply sample to column in a collection tube.</td></tr>
 <tr><td>↓ 15,000 rpmで1分間遠心し、ろ液を捨てる</td><td>&nbsp;</td><td>↓ Centrifuge for 1 minute at 15,000 rpm and discard flow-through.</td></tr>
-<tr><td>↓ 500 μlのBuffer QGをカラムに加えね残りのゲルを溶かす</td><td>&nbsp;</td><td>↓ Add 500 μl of Buffer QG to column to dissolve residual agarose.</td></tr>
+<tr><td>↓ 500 μlのBuffer QGをカラムに加え、残りのゲルを溶かす</td><td>&nbsp;</td><td>↓ Add 500 μl of Buffer QG to column to dissolve residual agarose.</td></tr>
 <tr><td>↓ 15,000 rpmで1分間遠心し、ろ液を捨てる</td><td>&nbsp;</td><td>↓ Centrifuge for 1 minute at 15,000 rpm and discard flow-through.</td></tr>
 <tr><td>↓ 750 μlのwash Buffer PEをカラムに加える</td><td>&nbsp;</td><td>↓ Add 750 μl of wash Buffer PE to column.</td></tr>
@@ Line 266: / Line 267: @@
 <span id="lig">'''ライゲーション'''</span></td><td width="20px">&nbsp;</td><td width="340px">
 <span id="ligeng">'''DNA ligation'''</span></td></tr>
-<tr><td>[https://www.roche-applied-science.com/servlet/RCProductDisplay?storeId=10202&catalogId=10202&langId=-1&countryId=us&forCountryId=us&productId=3.6.14.2.2.3 Rapid DNA Ligation Kit]を使用する</td><td>&nbsp;</td><td>We use [https://www.roche-applied-science.com/servlet/RCProductDisplay?storeId=10202&catalogId=10202&langId=-
+<tr><td>[https://www.roche-applied-science.com/servlet/RCProductDisplay?storeId=10202&catalogId=10202&langId=-1&countryId=us&forCountryId=us&productId=3.6.14.2.2.3 Rapid DNA Ligation Kit]を使用する</td><td>&nbsp;</td><td>We use [https://www.roche-applied-science.com/servlet/RCProductDisplay?storeId=10202&catalogId=10202&langId=-1&countryId=us&forCountryId=us&productId=3.6.14.2.2.3 Rapid DNA Ligation Kit].</td></tr>
-&countryId=us&forCountryId=us&productId=3.6.14.2.2.3 Rapid DNA Ligation Kit].</td></tr>
+<tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td>&nbsp;</td><td>↓ Mix the reagent according to the following components.</td></tr>
-<tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td>&nbsp;</td><td>>↓ Mix the reagent according to the following components.</td></tr>
 <tr><td><table border=1 width="300px"><tr><td align=center width="200px">5 x DNA dilution buffer</td><td align=right width="100px">2 μl</td></tr>
 <tr><td align=center>ベクター(カット・CHIP処理後)</td><td align=right>0.25-1 μl</td></tr>
 <tr><td align=center>インサート DNA</td><td align=right>0.5-6.5 μl</td></tr>
-<tr><td align=center>ddH2O</td><td align=right>全量が10 μlになるように調整する</td></tr></table></td><td>&nbsp;</td><td><table border=1 wight="300px"><tr><td wight="200px" align=center>5x DNA dilution buffer</td><td wight="100px" align=right>2 μl</td></tr>
+<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>全量が10 μlになるように調整する</td></tr></table></td><td>&nbsp;</td><td><table border=1 wight="300px"><tr><td wight="200px" align=center>5x DNA dilution buffer</td><td wight="100px" align=right>2 μl</td></tr>
 <tr><td align=center>Vector(cut and CHIP treated)</td><td align=right>0.25-1 μl</td></tr>
 <tr><td align=center>Insert DNA</td><td align=right>0.5-6.5 μl</td></tr>
-<tr><td align=center>ddH2O</td><td align=right>To final volume of 10 μl</td></tr></table></td></tr>
+<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>To final volume of 10 μl</td></tr></table></td></tr>
 <tr><td>↓ よく混ぜる</td><td>&nbsp;</td><td>↓ Mix well.</td></tr>
 <tr><td>↓ 10 μlの2 x rapid ligation bufferと1 μlのligaseを加える</td><td>&nbsp;</td><td>↓ Add 10 μl of
@@ Line 291: / Line 291: @@
 </td></tr>
+<tr><td>
+<table width="700px" align=center>
+<tr><td width="340px">
+<span id="com">'''コンピテント細胞の作製'''</span></td><td width="20px">&nbsp;</td><td width="340px">
+<span id="comeng">'''Preparing chemically competent cells'''</span></td></tr>
+<tr><td>↓ <I>E.coli</I>(DH5 Alpha)をLBプレートにまき、37 °Cで一晩培養する</td><td>&nbsp;</td><td>↓ Streak <I>E.coli</I> (DH5 Alpha) to LB plate and cultivate overnight in 37 °C.</td></tr>
+<tr><td>↓ シングルコロニーをピックアップして、2 mlのSOB培地で37 °Cで6時間培養する</td><td>&nbsp;</td><td>↓ Pick up a single colony from plate and cultivate 2 ml of SOB medium for 6 hours in 37 °C.</td></tr>
+<tr><td>↓ 培養液を250 mlのSOB培地に加える</td><td>&nbsp;</td><td>↓ Add 2 ml of the culture 250 ml of SOB medium.</td></tr>
+<tr><td>↓ 遠心しながら、37 °Cで吸光度(OD600)が0.4-0.8になるまで培養する</td><td>&nbsp;</td><td>↓ Cultivate at 37°C with agitation until the Optical Density (OD600) reaches 0.4-0.8.</td></tr>
+<tr><td>↓ 培養液を50 mlの遠心チューブに移し、氷上で10分間冷やす</td><td>&nbsp;</td><td>↓ Transfer the culture into 50 ml centrifuge tube and incubate the culture on ice for 10 minutes.</td></tr>
+<tr><td>↓ 4,500 rpm、4 °Cで10分間遠心し、上清を捨てる</td><td>&nbsp;</td><td>↓ Centrifuge for 10 minutes at 4,500 rpm in 4 °C and discard the supernatant.</td></tr>
+<tr><td>↓ 84 mlの氷冷した[http://openwetware.org/wiki/TB_buffer TB Buffer]を加えて、懸濁する</td><td>&nbsp;</td><td>↓ Add 84 ml of refrigerated [http://openwetware.org/wiki/TB_buffer TB Buffer] and resuspend.</td></tr>
+<tr><td>↓ 氷上で10分間冷やす</td><td>&nbsp;</td><td>↓ Incubate on ice for 10 minutes.</td></tr>
+<tr><td>↓ 4,500 rpm、4 °Cで10分間遠心し、上清を捨てる</td><td>&nbsp;</td><td>↓ Centrifuge for 10 minutes at 4,500 rpm in 4 °C and discard the supernatant.</td></tr>
+<tr><td>↓ 20 mlの氷冷した[http://openwetware.org/wiki/TB_buffer TB Buffer]を加えて、懸濁する</td><td>&nbsp;</td><td>↓ Add 20 ml of refrigerated [http://openwetware.org/wiki/TB_buffer TB Buffer] and resuspend.</td></tr>
+<tr><td>↓ 1.4 mlの7% DMSO(1.4 mlのDMSOに18.6 mlのTB Bufferを加えたもの)を加える</td><td>&nbsp;</td><td>↓ Add 1.4 ml of 7% DMSO(1.4 ml of DMSO to 18.6 ml of TB).</td></tr>
+<tr><td>↓ 氷上で10分間冷やす</td><td>&nbsp;</td><td>↓ Incubate on ice for 10 minutes.</td></tr>
+<tr><td>↓ 300 μlずつ分注する</td><td>&nbsp;</td><td>↓ Aliquot 300 μl of the cells.</td></tr>
+<tr><td>↓ 液体窒素で冷凍する</td><td>&nbsp;</td><td>↓ Freeze the cells in liquid nitrogen.</td></tr>
+<tr><td>↓ -80 °Cで保存する</td><td>&nbsp;</td><td>↓ Store at -80 °C.</td></tr>
+</table>
+</td></tr>
+<tr><td>
+<div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div>
+</td></tr>
+<tr><td>
+<table width="700px" align=center>
+<tr><td width="340px">
+<span id="seq">'''シークエンス'''</span></td><td width="20px">&nbsp;</td><td width="340px"><span id="seqeng">'''Sequencing'''</span></td></tr>
+<tr><td>PCR</td><td>&nbsp;</td><td>PCR</td></tr>
+<tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td>&nbsp;</td><td>↓ Mix the reagent according to the following components.</td></tr>
+<tr><td><table border=1 width="300px"><tr><td width="100px" align=center>H<sub>2</sub>O</td><td width="200px">全量10 μlになるように調整する</td></tr><tr><td align=center>Premix</td><td>2 μl</td></tr><tr><td align=center>5 x sequence buffer</td><td>1 μl</td></tr><tr><td align=center>Primer(1 μM)</td><td>1.6 μl</td></tr><tr><td align=center>プラスミド DNA</td><td>DNA量が100-250 ngになるように調整する</td></tr><tr><td>&nbsp;</td><td>全量 10 μl</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td width="100px" align=center>H<sub>2</sub>O</td><td width="200px">Adjust to become total 10 μl</td></tr><tr><td align=center>Premix</td><td>2 μl</td></tr><tr><td align=center>5 x sequence buffer</td><td>1 μl</td></tr>
+<tr><td align=center>Primer(1 μM)</td><td>1.6 μl</td></tr><tr><td align=center>Plasmid DNA</td><td>Adjust for the amount of DNA to become about 100-250 ng.</td></tr><tr><td>&nbsp;</td><td>10 μl system</td></tr></table></td></tr>
+<tr><td>↓ PCRのプログラム設定</td><td>&nbsp;</td><td>↓ PCR program</td></tr>
+<tr><td><table border=1 width="300px"><tr><td width="50%" align=center>Start</td><td>96 °C、1分</td></tr>
+<tr><td align=center rowspan=3>Cycle x 30</td><td>96 °C、10秒</td></tr><tr><td>50 °C、5秒</td></tr>
+<tr><td>60 °C、4分</td></tr><tr><td align=center>End</td><td>4 °Cで保つ</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td align=center width="100px">Start</td><td width="200px">96 °C for 1 minute.</td></tr><tr><td align=center rowspan=3>Cycle x 30</td><td width=>96 °C for 10 seconds. </td></tr><tr><td>50 °C for 5 seconds.</td></tr><tr><td>60 °C for 4 minutes.</td></tr><tr><td align=center>End</td><td>Keep at 4 °C forever.
+</td></tr></table></td></tr>
+<tr><td>エタノール沈殿</td><td>&nbsp;</td><td>EtOH precipitation</td></tr>
+<tr><td>↓ PCRの後、1.5 mlチューブに移す</td><td>&nbsp;</td><td>↓ Remove 1.5 ml tube after PCR.</td></tr>
+<tr><td>↓ 1.5 μlの3 M 酢酸ナトリウムを加える</td><td>&nbsp;</td><td>↓ Add 1.5 μl of 3 M CH3COONa.</td></tr>
+<tr><td>↓ 氷上で15分間冷やす</td><td>&nbsp;</td><td>↓ Incubate on ice for 15 minutes.</td></tr>
+<tr><td>↓ 31.5 μlの2-プロパノールと7 μlのH<sub>2</sub>Oを加える</td><td>&nbsp;</td><td>↓ Add 31.5 μl of 2-propanol and 7 μl of H<sub>2</sub>O.</td></tr>
+<tr><td>↓ 15,000 rpm、4 °Cで20分間遠心し、上清を捨てる</td><td>&nbsp;</td><td>↓ Centrifuge for 20 minutes at 15,000 rpm in 4 °C and discard the supernatant.</td></tr>
+<tr><td>↓ 50 μlの70% エタノールを加える.</td><td>&nbsp;</td><td>↓ Add 50 μl of 70% EtOH.</td></tr>
+<tr><td>↓ 15,000 rpm、4 °Cで20分間遠心し、上清を捨てる</td><td>&nbsp;</td><td>↓  Centrifuge for 20 minutes at 15,000 rpm in 4 °C and discard the supernatant.</td></tr>
+<tr><td>↓ 乾燥させる(よく乾かす)</td><td>&nbsp;</td><td>↓ Dry up.(Dry it well.)</td></tr>
+<tr><td>↓ 15 μlのHi-Di formamideに溶かす</td><td>&nbsp;</td><td>↓ Dissolve in 15 μl of Hi-Di formamide.</td></tr>
+<tr><td>↓ 95 °Cで2分加熱する</td><td>&nbsp;</td><td>↓ Incubate for 2 minutes at 95 °C.</td></tr>
+<tr><td>↓ 氷上で冷やす(急冷)</td><td>&nbsp;</td><td>↓ Incubate on ice(rapid cooling).</td></tr>
+<tr><td>↓ 0.5 mlチューブの蓋を切り取る</td><td>&nbsp;</td><td>↓ Cut out the cap of the 0.5 ml tube.</td></tr>
+<tr><td>↓ サンプルを1.5 mlチューブから0.5 mlチューブに移す</td><td>&nbsp;</td><td>↓ Remove the sample in the 1.5 ml tube to 0.5 ml tube.</td></tr>
+<tr><td>↓ シークエンス用の蓋をする</td><td>&nbsp;</td><td>↓ Do the cap for the sequencing.</td></tr>
+<tr><td>↓ シークエンサーに入れる</td><td>&nbsp;</td><td>↓ Take the sample in to sequencer.</td></tr>
+</table>
+</td></tr>
+<tr><td>
+<div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div>
+</td></tr>
+<tr><td>
+<table width="700px" align=center>
+<tr><td width="340px"><span id="gly">'''グリセロールストック'''</span></td><td width="20px">&nbsp;</td><td width="340px">
+<span id="glyeng">'''Glycerol stock'''</span></td></tr>
+<tr><td>↓ <I>E.coli</I> (DH5 Alpha)をLBプレートにまき、37 °Cで一晩培養する</td><td>&nbsp;</td><td>↓ Streak <I>E.coli</I> (DH5 Alpha) to LB plate and cultivate overnight in 37 °C.</td></tr>
+<tr><td>↓ シングルコロニーをピックアップして、2 mlのLB培地で37 °Cで一晩培養する</td><td>&nbsp;</td><td>↓ Pick up a single colony from plate and cultivate 2 ml of LB medium overnight in 37 °C.</td></tr>
+<tr><td>↓ 1.5 mlチューブ内で500 μlの培養液を500 μLの50%グリセロールに加える</td><td>&nbsp;</td><td>↓ Add 500 μl of an overnight culture to 500 μl of 50% glycerol in a 2 mL screw top tube.</td></tr>
+<tr><td>↓ -80 °Cでグリセロールストックを冷凍する</td><td>&nbsp;</td><td>↓ Freeze the glycerol stock tube at -80 °C.</td></tr>
+</table>
+</td></tr>
+<tr><td>
+<div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div>
+</td></tr>
+<tr><td>
+<table width="700px" align=center>
+<tr><td width="340px"><span id="lb">'''LB培地'''</span></td><td width="20px">&nbsp;</td><td width="340px">
+<span id="lbeng">'''LB culture medium'''</span></td></tr>
+<tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td>&nbsp;</td><td> ↓Mix the reagent according to the following components.</td></tr>
+<tr><td><table border=1 width="300px"><tr><td width="200px" align=center>Bacto
+Tryptone</td><td align=right>10 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5 g</td></tr>
+<tr><td align=center>NaCl</td><td align=right>1 g</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td width="200px" align=center>Bacto Tryptone</td><td align=right>10 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5 g</td></tr>
+<tr><td align=center>NaCl</td><td align=right>1 g</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td></tr>
+<tr><td>↓ 121 °Cで20分間オートクレーブする</td><td>&nbsp;</td><td>↓ Autoclave for 20 minutes at 121 °C.</td></tr>
+<tr><td>LBプレートを作るときは、20 gの寒天を加え、オートクレーブ後、20 mlずつプレートに温かい培地を注ぐ</td><td>&nbsp;</td><td>If you make LB plates, add 20 g of agar and after autoclave pour 20 ml of warm media into each plate.</td></tr>
+<tr><td>抗生物質入りのプレートを作るときは、オートクレーブ後、冷めた培地に抗生物質を加える</td><td>&nbsp;</td><td>If you make LB plates with the antibiotic, after autoclave add the antibiotic to the medium cooled.</td></tr>
+</table>
+</td></tr>
+<tr><td>
+<div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div>
+</td></tr>
+<tr><td>
+<table width="700px" align=center>
+<tr><td width="340px"><span id="yt">'''2 x YT培地'''</span></td><td width="20px">&nbsp;</td><td width="340px">
+<span id="yteng">'''2 x YT culture medium'''</span></td></tr>
+<tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td>&nbsp;</td><td>↓ Mix the reagent according to the following components.</td></tr>
+<tr><td><table border=1 width="300px"><tr><td width="200px" align=center>Bacto Tryptone</td><td align=right>16 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>10 g</td></tr><tr><td align=center>NaCl</td><td align=right>5 g</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td width="200px" align=center>Bacto Tryptone</td><td align=right>16 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>10 g</td></tr>
+<tr><td align=center>NaCl</td><td align=right>5 g</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td></tr>
+<tr><td>↓ 121 °Cで20分間オートクレーブする</td><td>&nbsp;</td><td>↓ Autoclave for 20 minutes at 121 °C.</td></tr>
+</table>
+</td></tr>
+<tr><td>
+<div align="right"><span style="font-ize:10pt;">[[#Contents|>>back to Contents]]</span></div>
+</td></tr>
+<tr><td>
+<table width="700px" align=center>
+<tr><td width="340px"><span id="sob">'''SOB培地'''</span></td><td width="20px">&nbsp;</td><td width="340px">
+<span id="sobeng">'''SOB culture medium'''</span></td></tr>
+<tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td>&nbsp;</td><td>↓ Mix the reagent according to the following components.</td></tr>
+<tr><td><table border=1 width="300px"><tr><td align=center width="200px">Bacto Tryptone</td><td align=right width="100px">20 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5g</td></tr><tr><td align=center>5M NaCl</td><td align=right>2ml</td></tr><tr><td align=center>2M KCl</td><td align=right>1.25 ml</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td align=center width="200px">Bacto Tryptone</td><td align=right width="100px">20 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5g</td></tr><tr><td align=center>5M NaCl</td><td align=right>2ml</td></tr>
+<tr><td align=center>2M KCl</td><td align=right>1.25 ml</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td></tr>
+<tr><td>↓ 121 °Cで20分間オートクレーブする</td><td>&nbsp;</td><td>↓ Autoclave for 20 minutes at 121 °C.</td></tr>
+<tr><td>↓ 10 mlの2M Mg solution (1 M MgSO<sub>4</sub>・7H<sub>2</sub>O +1 M MgCl<sub>2</sub>・6H<sub>2</sub>O)を加える</td><td>&nbsp;</td><td>↓ Add 10 ml of 2M Mg solution (1 M MgSO<sub>4</sub>・7H<sub>2</sub>O +1 M MgCl<sub>2</sub>・6H<sub>2</sub>O).</td></tr>
+</table>
+</td></tr>
+<tr><td>
+<div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div>
+</td></tr>
+<tr><td>
+<table width="700px" align=center>
+<tr><td width="340px"><span id="soc">'''SOC培地'''</span></td><td width="20px">&nbsp;</td><td width="340px">
+<span id="soceng">'''SOC culture medium'''</span></td></tr>
+<tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td>&nbsp;</td><td>↓ Mix the reagent according to the following components.</td></tr>
+<tr><td><table border=1 width="300px"><tr><td align=center width="200px">Bacto Tryptone</td><td align=right width="100px">20 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5g</td></tr><tr><td align=center>5M NaCl</td><td align=right>2ml</td></tr><tr><td align=center>2M KCl</td><td align=right>1.25 ml</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td align=center width="200px">Bacto Tryptone</td><td align=right width="100px">20 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5g</td></tr><tr><td align=center>5M NaCl</td><td align=right>2ml</td></tr><tr><td align=center>2M KCl</td><td align=right>1.25 ml</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td></tr>
+<tr><td>↓ 121 °Cで20分間オートクレーブする</td><td>&nbsp;</td><td>↓ Autoclave for 20 minutes at 121 °C.</td></tr>
+<tr><td>↓ 10 mlの2M Mg solution (1 M MgSO<sub>4</sub>・7H<sub>2</sub>O +1 M MgCl<sub>2</sub>・6H<sub>2</sub>O)を加える</td><td>&nbsp;</td><td>↓ Add 10 ml of 2M Mg solution (1 M MgSO<sub>4</sub>・7H<sub>2</sub>O +1 M MgCl<sub>2</sub>・6H<sub>2</sub>O).</td></tr>
+<tr><td>↓ 培地が50 °C以下に冷めた後、20 mlの滅菌済みの20%グルコース溶液を加える</td><td>&nbsp;</td><td>↓ After cooling medium to less than 50°C, add 20 ml filter sterilized 20% glucose solution.</td></tr>
+</table>
+</td></tr>
+<tr><td>
+<div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div>
+</td></tr>
 </table>
 {{Template:KIT-Kyoto-1}}