Team:KIT-Kyoto/Protocol

From 2010.igem.org

(Difference between revisions)

Revision as of 22:08, 23 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		↓ 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.

>>back to Contents

アルカリミニプレップ

DNA miniprep

<tdalign=right>25 mM Tris-HCl (pH 8.0)</td> <tr><td align=center>Solution II</td><td align=right>0.2 N NaOH</td></tr><tr><td> </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> </td><td align=right>1.8 M 酢酸</td></tr></table></td><td> </td><td>

Solution I	50 mM グルコース (MW 180)
	10 mM EDTA(pH 8.0)

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

</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> </td><td>↓ Streak E.coli 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> </td><td>↓ Pick up a single colony from plate.</td></tr> <tr><td>↓ 2 mlのLB培地で37 °Cで一晩振とう培養する</td><td> </td><td>↓ Cultivate in 2 ml LB medium overnight in 37 °C, shaking.</td></tr> <tr><td>↓ 1.5 mlの培養液を1.5 mlチューブにうつす</td><td> </td><td>↓ Pipet 1.5 ml of overnight culture into a 1.5 ml tube. </td></tr> <tr><td>↓ 15,000 rpm、4 °Cで1分間遠心し、上清を捨てる</td><td> </td><td>↓ Centrifuge for 1 minute at 15,000 rpm in 4 °C and discard the supernatant.</td></tr> <tr><td>↓ 100 μlの氷冷したSolution Iを沈殿に加え、懸濁する</td><td> </td><td>↓ Add 100 μl of refrigerated Solution I to the pellet and vortex to resuspend.</td></tr> <tr><td>↓ 200 μlのSolution IIを加え、混ぜる、ボルテックスは使用しない</td><td> </td><td>↓ Add 200 μl of Solution II and invert gently to mix. Do not vortex.</td></tr> <tr><td>↓ 氷上で5分間冷やす</td><td> </td><td>↓ Incubate on ice for 5 minutes.</td></tr> <tr><td>↓ 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</td><td> </td><td>↓ Add 150 μl of refrigerated Solution III and invert gently to mix. Do not vortex.</td></tr> <tr><td>↓ 氷上で5分間冷やす</td><td> </td><td>↓ Incubate on ice for 5 minutes.</td></tr> <tr><td>↓ 15,000 rpm、4 °Cで5分間遠心する</td><td> </td><td>↓ Centrifuge for 5 minutes at 15,000 rpm in 4 °C.</td></tr> <tr><td>↓ 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</td><td> </td><td>↓ Carefully pipet 400 μl of the clean supernatant into a new tube.</td></tr> <tr><td>↓ 900 μlの2-プロパノールを加え、混ぜる</td><td> </td><td>↓ Add 900 μl of 2-propanol and mix.</td></tr> <tr><td>↓ 2分間室温で放置する</td><td> </td><td>↓ Incubate for 2 minutes in room temperature.</td></tr> <tr><td>↓ 15,000 rpm、4 °Cで10分間遠心し、上清を捨てる</td><td> </td><td>↓ Centrifuge for 10 minutes at 15,000 rpm in 4 °C and discard supernatant.</td></tr> <tr><td>↓ 1 mlの70%エタノールを加える</td><td> </td><td>↓ Add 1 ml 70% EtOH to the pellet.</td></tr> <tr><td>↓ 15,000 rpm、4 °Cで2分間遠心し、上清を捨てる</td><td> </td><td>↓ Centrifuge for 2 minutes at 15,000 rpm in 4 °C and discard supernatant.</td></tr> <tr><td>↓ 沈殿を10分～15分乾かす</td><td> </td><td>↓ Dry the pellet for 10-15 minutes. </td></tr> <tr><td>↓ プラスミドDNAを30 μlのRNaseのはいったTEに溶かす</td><td> </td><td>↓ Resuspend the plasmid DNA in 30 μl of TE with RNase. </td></tr></table>

</td></tr> <tr><td>

>>back to Contents

</td></tr>

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
	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.
	Keep at 4 °C forever.

</td></tr> <tr><td>

>>back to Contents

</td></tr>

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=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.

</td></tr> <tr><td>

>>back to Contents

</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>10 mM EDTA(pH 8.0)</td></tr><tr><td>&nbsp;</td><td>25 mM Tris-HCl (pH 8.0)</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><tdalign=right>25 mM Tris-HCl (pH 8.0)</td></tr>
-<tr><td align=center>Solution II</td><td>0.2 N NaOH</td></tr><tr><td>&nbsp;</td><td>1% SDS</td></tr><tr><td align=center>Solution III</td><td>3 M 酢酸カリウム</td></tr><tr><td>&nbsp;</td><td>1.8 M 酢酸</td></tr></table></td><td>&nbsp;</td><td><table
+<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
-border=1 width="300px"><tr><td width="100px" align=center>Solution I</td><td width="200px">50 mM glucose (MW 180)</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>&nbsp;</td><td>10 mM EDTA(pH 8.0)</td></tr><tr><td>&nbsp;</td><td>25 mM Tris-HCl (pH 8.0)</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 align=center>Solution II</td><td>0.2 N NaOH</td></tr><tr><td>&nbsp;</td><td>1% SDS</td></tr><tr><td align=center>Solution III</td><td>3 M potassium acetate</td></tr><tr><td>&nbsp;</td><td>1.8 M acetic acid</td></tr></table></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>↓ 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 110: / Line 110: @@
 <tr><td>↓ 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</td><td>&nbsp;</td><td>↓ Design primers with software programs.</td></tr><tr><td>[http://ginkgobioworks.com/cgi/primer.cgi>>Ginkgo BioWorks]</td><td>&nbsp;</td><td>[http://ginkgobioworks.com/cgi/primer.cgi>>Ginkgo BioWorks]</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>Primer Mix</td><td width="100px">1.5 μl</td></tr>
+<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>0.5 μl</td></tr><tr><td align=center>10 x PCR Buffer for KOD-Plus-</td><td>5 μl</td></tr><tr><td align=center>2 mM dNTPs</td><td>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>4 μl</td></tr><tr><td align=center>ddH2O</td><td>33 μl</td></tr><tr><td align=center>KOD-Plus-</td><td>1 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>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">1.5 μl</td></tr><tr><td align=center>Template DNA</td><td>0.5 μl</td></tr><tr><td align=center>10 x PCR Buffer for KOD-Plus-</td><td>5 μl</td></tr><tr><td align=center>2 mM dNTPs</td><td>5 μl</td></tr><tr><td align=center>2 mM MgSO4</td><td>4 μl</td></tr><tr><td align=center>ddH2O</td><td>33 μl</td></tr><tr><td align=center>KOD-Plus-</td><td>1 μl</td></tr><tr><td>&nbsp;</td><td>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 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>↓ 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">94 °C、2分</td></tr><tr><td align=center>Cycle x 35</td><td>94 °C、15秒 (熱変性)</td></tr><tr><td>&nbsp;</td><td>(Tm-10) °C、0.5分 (アニーリング)</td></tr><tr><td>&nbsp;</td><td>68 °C、1 kb/min (伸長)</td></tr><tr><td align=center>End</td><td>68 °C、2分</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>Cycle x 35</td><td align=right>94 °C、15秒 (熱変性)</td></tr><tr><td>&nbsp;</td><td align=right>(Tm-10) °C、0.5分 (アニーリング)</td></tr><tr><td>&nbsp;</td><td align=right>68 °C、1 kb/min (伸長)</td></tr><tr><td align=center>End</td><td align=right>68 °C、2分</td></tr>
-<tr><td>&nbsp;</td><td>4 °Cで保存</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td width="60px" align=center>Start</td><td width="240px">94 °C for 2 minutes.</td></tr><tr><td align=center>Cycle x 35</td><td>94 °C for 15 seconds. (denaturing)</td></tr><tr><td>&nbsp;</td><td>(Tm-10) °C for 0.5 minutes. (annealing)</td></tr><tr><td>&nbsp;</td><td>68 °C for 1 kb/min. (extension)</td></tr><tr><td align=center>End</td><td>68 °C for 2 minutes.</td></tr><tr><td>&nbsp;</td><td>Keep at 4 °C forever.</td></tr></table></td></tr>
+<tr><td>&nbsp;</td><td align=right>4 °Cで保存</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px"><tr><td width="60px" align=center>Start</td><td width="240px" align=right>94 °C for 2 minutes.</td></tr><tr><td align=center>Cycle x 35</td><td align=right>94 °C for 15 seconds. (denaturing)</td></tr><tr><td>&nbsp;</td><td align=right>(Tm-10) °C for 0.5 minutes. (annealing)</td></tr><tr><td>&nbsp;</td><td align=right>68 °C for 1 kb/min. (extension)</td></tr><tr><td align=center>End</td><td align=right>68 °C for 2 minutes.</td></tr><tr><td>&nbsp;</td><td align=right>Keep at 4 °C forever.</td></tr></table></td></tr>
 </table>
 </td></tr>
@@ Line 131: / Line 131: @@
 <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="120px" align=center>10 x Buffer</td><td rowspan=3 width="180px">2 μl (全量の10分の1)</td></tr>
+<tr><td width="120px" align=center>10 x Buffer</td><td rowspan=3 width="180px" align=right>2 μl (全量の10分の1)</td></tr>
 <tr><td frame="vsides" align=center>
 :[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]</td></tr>
@@ Line 144: / Line 144: @@
 <tr><td frame=vsides 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>ddH2O</td><td align=right>全量が20 μlになるように調整する</td></tr>
-<tr><td>&nbsp;</td><td>全量 20 μl</td></tr></table></td><td>&nbsp;</td><td><table border=1 width="300px">
+<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>
+<tr><td width="120px" align=center>10 x Buffer</td><td rowspan=3 width="180px" align=right>2 µl (1/10 of total)</td></tr>
 <tr><td frame=vsides align=center>
 :[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]</td></tr>
 <tr><td frame=vsides align=center>
 :[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]</td></tr>
-<tr><td align=center>DNA solution</td><td>Adjust for the amount of DNA to become 1 μg.</td></tr>
+<tr><td align=center>DNA solution</td><td align=right>Adjust for the amount of DNA to become 1 μg.</td></tr>
-<tr><td align=center>Restriction enzyme</td><td rowspan=5>Adjust to become 3 units.</td></tr>
+<tr><td align=center>Restriction enzyme</td><td rowspan=5 align=right>Adjust to become 3 units.</td></tr>
 <tr><td frame=vsides align=center>
 :[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 <I>Eco</I>R I]</td></tr>
@@ Line 161: / Line 161: @@
 <tr><td frame=vsides 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>ddH2O</td><td align=right>Adjust to become total 20 μl.</td></tr>
-<tr><td>&nbsp;</td><td>20 μl system</td></tr></table></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>
 </table>