http://w.atwiki.jp/transfection/ transfection ja 2007-08-07T14:57:27+09:00 1186466247 https://w.atwiki.jp/transfection/pages/2.html メニュー -[[トップページ]] -[[日々の記録>http://pathol1.cocolog-nifty.com/]] ---- #calendar -[[@ウィキ ガイド>http://atwiki.jp/guide/]] -[[@wiki 便利ツール >http://atwiki.jp/tools/]] -[[@wiki>http://atwiki.jp]] // リンクを張るには "[" 2つで文字列を括ります。 // ">" の左側に文字、右側にURLを記述するとリンクになります **更新履歴 #recent(20) 2007-08-07T14:57:27+09:00 1186466247 https://w.atwiki.jp/transfection/pages/1.html -[[PCR]] -[[エタノール沈殿]] -[[電気泳動]] -[[DNA精製]] -[[制限酵素処理]] -[[ゲル濾過]] -[[DNA Ligation]] -[[Transformation]] -[[コンピテントセルのピックアップ]] 2007-08-07T14:55:42+09:00 1186466142 https://w.atwiki.jp/transfection/pages/25.html <p><strong>所要</strong>　時間　</p> <p><strong>実作業</strong>　分</p> <p><strong>手順</strong></p> <ol> <li>1plateあたりplasmid 0.3mlに対し、制限酵素(BamHI)反応液7.0mlを 加える。</li> <li>24+1 plate＝25plate分のマスターmixtureを作成（1plate分はピペットのロス）</li> <li>24plateにまく(テラサキプレート)</li> <li>各plateにplasmid3mlを加え、37℃ 1時間30分incubate</li> <li>[[電気泳動]]（0.9%アガロース）によりinsert check</li> </ol> <h4>資料</h4> <ol> <li>反応液の組成</li> <li style="list-style-type: none"> <table style= "border-right: medium none; border-top: medium none; border-left: medium none; border-bottom: medium none; border-collapse: collapse" cellspacing="0" cellpadding="0" border="1"> <tbody> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">内容(DMSO+)</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">量(㎕)</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <p style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">内容<span>(DMSO-)</span></span></p> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">量(㎕)</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">Template</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5 (100ng)</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">Template</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5 (100ng)</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Forward primer</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Forward primer</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Reverse primer</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Reverse primer</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">2mM dNTP</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">dNTP/HiFi buffer premix</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">10</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">25mM MgSO4</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">2</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">x10 KOD plus buffer</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">HiFi</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">0.35</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">KOD plus</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">1</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">KOD plus</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">10% DMSO</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">25</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">H2O</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">10.5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">H2O</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">37.65</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">50</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">50</span></div> </td> </tr> </tbody> </table> </li> </ol> 2007-07-23T16:19:29+09:00 1185175169 https://w.atwiki.jp/transfection/pages/14.html <p><strong>所要</strong>　2時間　</p> <p><strong>実作業</strong>　30分</p> <p><strong>手順</strong></p> <ol> <li>チューブに反応液の調整（DMSOあり、なしの2種類の反応液）</li> <li>サーマルサイクラーへセット</li> </ol> <h4>資料</h4> <ol> <li>反応液の組成</li> <li style="list-style-type: none"> <table style= "border-right: medium none; border-top: medium none; border-left: medium none; border-bottom: medium none; border-collapse: collapse" cellspacing="0" cellpadding="0" border="1"> <tbody> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">内容(DMSO+)</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">量(㎕)</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <p style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">内容<span>(DMSO-)</span></span></p> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">量(㎕)</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">Template</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5 (100ng)</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">Template</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5 (100ng)</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Forward primer</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Forward primer</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Reverse primer</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Reverse primer</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">2mM dNTP</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">dNTP/HiFi buffer premix</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">10</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">25mM MgSO4</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">2</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">x10 KOD plus buffer</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">HiFi</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">0.35</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">KOD plus</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">1</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">KOD plus</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">10% DMSO</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">25</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">H2O</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">10.5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">H2O</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">37.65</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">50</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">50</span></div> </td> </tr> </tbody> </table> </li> </ol> <p><strong>KOD-plus-</strong><br> 東洋紡ライフサイエンス社製DNA polymerase</p> <p><strong>HiFi（Expand High Fidelity^PLUSPCR System）</strong><br> Rocheアプライドサイエンス社製DNA polymerase混合物</p> 2007-07-23T16:10:01+09:00 1185174601 https://w.atwiki.jp/transfection/pages/24.html <p><strong>所要</strong>　2時間　</p> <p><strong>実作業</strong>　30分</p> <p><strong>手順</strong></p> <ol> <li>チューブに番号を振る（24本作成）</li> <li>LB/ampプレートを新たに用意し、チューブの本数分領域分割し、番号を振る</li> <li>チューブにアンピシリン添加TB培地を700μずつ分注</li> <li>滅菌した爪楊枝で、プレートから1コロニーをピックアップ（ひっかく）。</li> <li>1つのチューブ内で爪楊枝を数回まわして、細胞を少し落とす（液体培養にする）</li> <li>新しいプレート内にも、チューブに対応する領域（番号）に細胞を植える</li> <li>24本のチューブ全てに対して、3.-5.の作業を行う</li> <li>37℃でovernightインキュベート（チューブは振盪させる。プレートは静置）</li> </ol> <p>コメント</p> <ul> <li>チューブが24本なのは、plasmid isolation system（KURABO社PI-24）にセットできる上限のため。</li> <li>プレートからコロニーをピックアップする場合、同時に2つのコロニーをひっかけないように注意。<br> （離れたコロニーを選んでピックアップするのが無難）</li> <li>プレートからまんべんなくコロニーをピックアップした方がよいという意見がある。</li> </ul> 2007-07-23T16:01:35+09:00 1185174095 https://w.atwiki.jp/transfection/pages/23.html <p><strong>所要</strong>　一晩　</p> <p><strong>実作業</strong>　10分</p> <p><strong>手順</strong></p> <ol> <li>Ligation反応液にコンピテントセル(SURE2) 100?を加える</li> <li>氷上で10分放置</li> <li>42℃で1分間加熱（heat shock）</li> <li>LB培地900?を添加</li> <li>37℃で30分間振盪</li> <li>軽く遠心（30秒ぐらい）して、余分な溶液（培地）を捨てる</li> <li>残った溶液でペレットを十分に溶解</li> <li>LB/ampプレートに溶液を滴下し、コンラージ棒で塗抹</li> <li>37℃でovernightインキュベート</li> </ol> <h4>コメント</h4> <ul> <li>heat shockは不要という人もいる。</li> </ul> 2007-07-23T15:53:48+09:00 1185173628 https://w.atwiki.jp/transfection/pages/22.html <p><strong>所要</strong>　3時間　</p> <p><strong>実作業</strong>　10分</p> <p><strong>手順</strong></p> <ol> <li>反応液の調整</li> <li>16℃で2,3時間反応</li> </ol> <h4>資料</h4> <ol> <li>反応液の組成 <table style="border-collapse: collapse" cellspacing="0" cellpadding="0" border="0"> <tbody> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">DNA</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">量(㎕)</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Insert (ゲル濾過したDNA)</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">4</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Vector (pCX4)</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">x5 ligase buffer</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">4</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">T4 DNA ligase</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">1</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">H2O</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">10.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">20</span></div> </td> </tr> </tbody> </table> </li> </ol> 2007-07-23T15:48:14+09:00 1185173294 https://w.atwiki.jp/transfection/pages/21.html <p><strong>所要</strong>　2時間　</p> <p><strong>実作業</strong>　30分</p> <p><strong>手順</strong></p> <ol> <li>1mlのシリンジに綿棒の綿をほぐしてつめる（目盛り0.1mlぐらいまで）</li> <li>0．25M NaCl/TE bufferに膨潤したセファデックスG50（Amersham社）を1mlまで注ぐ</li> <li>buffer 300㎕を6回注ぐ</li> <li>サンプル（<span>DNAとvector）を注ぐ</span></li> <li><span>buffer 250㎕を注いで、捨てる</span></li> <li><span>buffer 300㎕を注いで、濾過液をチューブに回収</span></li> </ol> <p><span><strong>コメント</strong></span></p> <ul> <li>bufferの間に空気が入ると、液が落ちていかないので注意。</li> </ul> 2007-07-23T15:44:36+09:00 1185173076 https://w.atwiki.jp/transfection/pages/15.html <p><strong>所要　</strong>約30分</p> <p><strong>実作業　</strong>1<font size="2">0分</font></p> <p><strong>手順</strong></p> <ol> <li>PCR産物に3M NaOAc 5㎕、100%エタノール125㎕加える</li> <li>室温で<span>5分程度放置</span></li> <li>15000回転(4℃)で15分間遠心</li> <li>上清を捨てる（マイクロピペットを使用）</li> <li>70%エタノールを100㎕程度加える</li> <li>15000回転(4℃)で3分間遠心</li> <li>上清を捨てる（マイクロピペットを使用）</li> <li>乾燥（<span>70℃に加熱しても良い）</span></li> </ol> <p><span><strong>コメント</strong></span></p> <ul> <li><span>ゲル濾過後など塩<span>(NaCl)が入った後のエタノール沈殿では、NaOACは不要。</span></span></li> <li><span><span>産物の量が少ない時は、<span>GlycoBlue(Ambion社) 1㎕添加して行う。</span></span></span></li> </ul> 2007-07-23T15:36:16+09:00 1185172576 https://w.atwiki.jp/transfection/pages/20.html <p><span lang="EN-US" style= "font-size: 10pt; font-family: 'ＭＳ Ｐゴシック'; mso-bidi-font-size: 12.0pt"><strong>所要　</strong></span><span lang="EN-US" style= "font-size: 10pt; font-family: 'ＭＳ Ｐゴシック'; mso-bidi-font-size: 12.0pt">一晩</span></p> <p class="MsoNormal" style= "margin: 0mm 0mm 0pt 20.25pt; text-indent: -20.25pt; mso-list: l8 level1 lfo1; tab-stops: list 20.25pt"> <span lang="EN-US" style= "font-size: 10pt; font-family: 'ＭＳ Ｐゴシック'; mso-bidi-font-size: 12.0pt"><strong>実作業　</strong>5</span><font size="2">分</font></p> <p class="MsoNormal" style= "margin: 0mm 0mm 0pt 20.25pt; text-indent: -20.25pt; mso-list: l8 level1 lfo1; tab-stops: list 20.25pt">  </p> <h4 class="MsoNormal" style= "margin: 0mm 0mm 0pt 20.25pt; text-indent: -20.25pt; mso-list: l8 level1 lfo1; tab-stops: list 20.25pt"> <font size="2">手順</font></h4> <ol> <li>反応液の調整</li> <li><span lang="EN-US" style= "font-size: 10pt; font-family: 'ＭＳ Ｐゴシック'; mso-bidi-font-size: 12.0pt"><span id="fck_dom_range_start_1185164029371_784"> <span id= "fck_dom_range_start_1185164030893_235">37℃でovernight反応</span></span></span></li> </ol> <h4><span lang="EN-US" style= "font-size: 10pt; font-family: 'ＭＳ Ｐゴシック'; mso-bidi-font-size: 12.0pt"><span id="fck_dom_range_start_1185164029371_784"> <span id="fck_dom_range_start_1185164030893_235">資料</span></span></span></h4> <ol> <li><span lang="EN-US" style= "font-size: 10pt; font-family: 'ＭＳ Ｐゴシック'; mso-bidi-font-size: 12.0pt"><span id="fck_dom_range_start_1185164029371_784"> <span id= "fck_dom_range_start_1185164030893_235">反応液の組成<br></span></span></span> <table style="border-collapse: collapse" cellspacing="0" cellpadding="0" border="0"> <tbody> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">DNA</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">量(㎕)</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">Vector</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">量(㎕)</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">Insert (CD10)</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">20</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">pCX4Puro</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">0.8(2㎍)</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">BamH1</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">BamH1</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">Mfe1</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">Mfe1</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">0.5</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">H2O</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">16.2</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">X10制限酵素buffer</span></div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">2.4</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">X10制限酵素buffer</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: windowtext 0.5pt solid; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">2.0</span></div> </td> </tr> <tr> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: windowtext 0.5pt solid; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style= "font-size: 10pt">23.4</span></div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"> </div> </td> <td style= "border-right: #d4d0c8; padding-right: 4.95pt; border-top: #d4d0c8; padding-left: 4.95pt; padding-bottom: 0mm; border-left: #d4d0c8; padding-top: 0mm; border-bottom: #d4d0c8; background-color: transparent" valign="top"> <div style="margin: 0mm 0mm 0pt"><span style="font-size: 10pt">20</span></div> </td> </tr> </tbody> </table> </li> </ol> <p> </p> <h4>コメント</h4> <ul> <li>環状DNAよりも直鎖状DNAは制限酵素処理の効率が悪い（時間がかかる）ので、一晩反応。</li> </ul> <p> </p> 2007-07-23T15:32:40+09:00 1185172360