鉴定PCR扩增产物的实验步骤

<p class="MsoNormal"><strong><span style="color: rgb(255, 0, 0);"><span style="font-family: 宋体; font-size: 9pt;">实验原理</span></span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">琼脂糖凝胶电泳鉴定</font>PCR<font face="宋体">扩增产物。（原理参照琼脂糖凝胶电泳检测</font><font face="Calibri">DNA</font><font face="宋体">实验）</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">本实验使用了公司的胶纯化试剂盒，该试剂盒是从琼脂糖凝胶中回收并纯化</font>DNA<font face="宋体">片段的试剂盒。试剂盒采用了独特的凝胶融解系统，结合</font><font face="Calibri">DNA</font><font face="宋体">制备膜技术，具有、快速、方便的特点，全套操作只需</font><font face="Calibri">30min</font><font face="宋体">便可完成。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">使用该试剂盒每次可纯化得到多至</font>10&micro;g<font face="宋体">的</font><font face="Calibri">DNA</font><font face="宋体">片段（</font><font face="Calibri">100bp</font><font face="宋体">～</font><font face="Calibri">30kb</font><font face="宋体">），回收率高达</font><font face="Calibri">50</font><font face="宋体">～</font><font face="Calibri">80%</font><font face="宋体">。本试剂盒回收纯化的</font><font face="Calibri">DNA</font><font face="宋体">片段纯度高，完整性好，可直接用于连接反应、</font><font face="Calibri">PCR</font><font face="宋体">扩增、</font><font face="Calibri">DNA</font><font face="宋体">测序等各种分子生物学实验。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">经胶纯化试剂盒回收的</font>DNA<font face="宋体">片段采用</font><font face="Calibri">-20</font><font face="宋体">℃低温保存，延缓</font><font face="Calibri">DNA</font><font face="宋体">的降解。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="color: rgb(255, 0, 0);"><span style="font-family: 宋体; font-size: 9pt;">实验步骤</span></span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">1.</span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"> <font face="宋体">使用</font><font face="Calibri">1</font><font face="宋体">&times;</font><font face="Calibri">TAE</font><font face="宋体">缓冲液制作琼脂糖凝胶，然后对目的</font><font face="Calibri">DNA</font><font face="宋体">进行琼脂糖凝胶电泳。（参照琼脂糖凝胶电泳检测</font><font face="Calibri">DNA</font><font face="宋体">实验）</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">2. </span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">在紫外灯下切出含有目的</font><font face="Calibri">DNA</font><font face="宋体">（约</font><font face="Calibri">600bp</font><font face="宋体">）的琼脂糖凝胶，用纸巾吸尽凝胶表面的液体。此时应注意尽量切除不含目的</font><font face="Calibri">DNA</font><font face="宋体">部分的凝胶，尽量减小凝胶体积，提高</font><font face="Calibri">DNA</font><font face="宋体">回收率。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="color: rgb(255, 102, 0);"><span style="font-family: 宋体; font-size: 9pt;"><font face="宋体">注意：</font></span></span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">切胶时请注意不要将</font>DNA<font face="宋体">长时间暴露于紫外灯下，以防止</font><font face="Calibri">DNA</font><font face="宋体">损伤。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">3. </span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">切碎胶块。胶块切碎后可以加快操作步骤</font><font face="Calibri">6</font><font face="宋体">的胶块融化时间，提高</font><font face="Calibri">DNA</font><font face="宋体">的回收率。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">4. <font face="宋体">称量胶块重量，计算胶块体积。计算胶块体积时，以</font><font face="Calibri">1mg=1&micro;L</font><font face="宋体">进行计算。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">5.</span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"> <font face="宋体">向胶块中加入胶块融化液</font><font face="Calibri">DR-I Buffer</font><font face="宋体">，</font><font face="Calibri">DR-I Buffer</font><font face="宋体">的加量方法如下：</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">① </span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">均匀混合后<font face="Calibri">75</font><font face="宋体">℃加热，融化胶块（低熔点琼脂糖凝胶只需在</font><font face="Calibri">45</font><font face="宋体">℃加热）。此时应间断振荡混合，使胶块充分融化（约</font><font face="Calibri">6</font><font face="宋体">～</font><font face="Calibri">10</font><font face="宋体">分钟）。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 102, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;"><font face="宋体">注意：</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">胶块一定要充分融化，否则将会严重影响</font>DNA<font face="宋体">的回收率。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">②</span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"> 向上述胶块融化液中加入<font face="Calibri">DR-I Buffer</font><font face="宋体">量的</font><font face="Calibri">1/2</font><font face="宋体">体积量的</font><font face="Calibri">DR-II Buffer</font><font face="宋体">，均匀混合。当分离小于</font><font face="Calibri">400 bp</font><font face="宋体">的</font><font face="Calibri">DNA</font><font face="宋体">片段时，应在此溶液中再加入终浓度为</font><font face="Calibri">20%</font><font face="宋体">的异丙醇。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">6.</span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"> <font face="宋体">将试剂盒中的</font><font face="Calibri">Spin Column</font><font face="宋体">安置于</font><font face="Calibri">Collection Tube</font><font face="宋体">上。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">7. </span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">将上述操作</font><font face="Calibri">7</font><font face="宋体">的溶液转移至</font><font face="Calibri">Spin Column</font><font face="宋体">中，</font><font face="Calibri">12000 rpm</font><font face="宋体">离心</font><font face="Calibri">1min</font><font face="宋体">，弃滤液。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 102, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;"><font face="宋体">注意：</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">如将滤液再加入</font>Spin Column<font face="宋体">中离心一次，可以提高</font><font face="Calibri">DNA</font><font face="宋体">的回收率。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">8. </span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">将</font><font face="Calibri">500 &micro;L</font><font face="宋体">的</font><font face="Calibri">Rinse A</font><font face="宋体">加入</font><font face="Calibri">Spin Column</font><font face="宋体">中，</font><font face="Calibri">12000 rpm</font><font face="宋体">离心</font><font face="Calibri">30s</font><font face="宋体">，弃滤液。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">9. </span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">将</font><font face="Calibri">700 &micro;L</font><font face="宋体">的</font><font face="Calibri">Rinse B</font><font face="宋体">加入</font><font face="Calibri">Spin Column</font><font face="宋体">中，</font><font face="Calibri">12000 rpm</font><font face="宋体">离心</font><font face="Calibri">30s</font><font face="宋体">，弃滤液。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">10. </span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">重复操作步骤</font><font face="Calibri">11</font><font face="宋体">。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">11.</span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"> <font face="宋体">将</font><font face="Calibri">Spin Column</font><font face="宋体">安置于新的</font><font face="Calibri">1.5 mL</font><font face="宋体">的离心管上，在</font><font face="Calibri">Spin Column</font><font face="宋体">膜的中央处加入</font><font face="Calibri">25 &micro;L</font><font face="宋体">的灭菌蒸馏水或</font><font face="Calibri">Elution Buffer</font><font face="宋体">，室温静置</font><font face="Calibri">1min</font><font face="宋体">。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>