鑒定PCR擴增產(chǎn)物的實驗步驟

<p class="MsoNormal"><strong><span style="color: rgb(255, 0, 0);"><span style="font-family: 宋體; font-size: 9pt;">實驗原理</span></span></strong><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">瓊脂糖凝膠電泳鑒定</font>PCR<font face="宋體">擴增產(chǎn)物。（原理參照瓊脂糖凝膠電泳檢測</font><font face="Calibri">DNA</font><font face="宋體">實驗）</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">本實驗使用了公司的膠純化試劑盒，該試劑盒是從瓊脂糖凝膠中回收并純化</font>DNA<font face="宋體">片段的試劑盒。試劑盒采用了獨特的凝膠融解系統(tǒng)，結合</font><font face="Calibri">DNA</font><font face="宋體">制備膜技術，具有、快速、方便的特點，全套操作只需</font><font face="Calibri">30min</font><font face="宋體">便可完成。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">使用該試劑盒每次可純化得到多至</font>10&micro;g<font face="宋體">的</font><font face="Calibri">DNA</font><font face="宋體">片段（</font><font face="Calibri">100bp</font><font face="宋體">～</font><font face="Calibri">30kb</font><font face="宋體">），回收率高達</font><font face="Calibri">50</font><font face="宋體">～</font><font face="Calibri">80%</font><font face="宋體">。本試劑盒回收純化的</font><font face="Calibri">DNA</font><font face="宋體">片段純度高，完整性好，可直接用于連接反應、</font><font face="Calibri">PCR</font><font face="宋體">擴增、</font><font face="Calibri">DNA</font><font face="宋體">測序等各種分子生物學實驗。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">經(jīng)膠純化試劑盒回收的</font>DNA<font face="宋體">片段采用</font><font face="Calibri">-20</font><font face="宋體">℃低溫保存，延緩</font><font face="Calibri">DNA</font><font face="宋體">的降解。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">實驗步驟</span></span></strong><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">使用</font><font face="Calibri">1</font><font face="宋體">&times;</font><font face="Calibri">TAE</font><font face="宋體">緩沖液制作瓊脂糖凝膠，然后對目的</font><font face="Calibri">DNA</font><font face="宋體">進行瓊脂糖凝膠電泳。（參照瓊脂糖凝膠電泳檢測</font><font face="Calibri">DNA</font><font face="宋體">實驗）</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">在紫外燈下切出含有目的</font><font face="Calibri">DNA</font><font face="宋體">（約</font><font face="Calibri">600bp</font><font face="宋體">）的瓊脂糖凝膠，用紙巾吸盡凝膠表面的液體。此時應注意盡量切除不含目的</font><font face="Calibri">DNA</font><font face="宋體">部分的凝膠，盡量減小凝膠體積，提高</font><font face="Calibri">DNA</font><font face="宋體">回收率。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;"><font face="宋體">注意：</font></span></span></strong><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">切膠時請注意不要將</font>DNA<font face="宋體">長時間暴露于紫外燈下，以防止</font><font face="Calibri">DNA</font><font face="宋體">損傷。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">切碎膠塊。膠塊切碎后可以加快操作步驟</font><font face="Calibri">6</font><font face="宋體">的膠塊融化時間，提高</font><font face="Calibri">DNA</font><font face="宋體">的回收率。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">稱量膠塊重量，計算膠塊體積。計算膠塊體積時，以</font><font face="Calibri">1mg=1&micro;L</font><font face="宋體">進行計算。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">向膠塊中加入膠塊融化液</font><font face="Calibri">DR-I Buffer</font><font face="宋體">，</font><font face="Calibri">DR-I Buffer</font><font face="宋體">的加量方法如下：</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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;">均勻混合后<font face="Calibri">75</font><font face="宋體">℃加熱，融化膠塊（低熔點瓊脂糖凝膠只需在</font><font face="Calibri">45</font><font face="宋體">℃加熱）。此時應間斷振蕩混合，使膠塊充分融化（約</font><font face="Calibri">6</font><font face="宋體">～</font><font face="Calibri">10</font><font face="宋體">分鐘）。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;"><font face="宋體">注意：</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">膠塊一定要充分融化，否則將會嚴重影響</font>DNA<font face="宋體">的回收率。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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;"> 向上述膠塊融化液中加入<font face="Calibri">DR-I Buffer</font><font face="宋體">量的</font><font face="Calibri">1/2</font><font face="宋體">體積量的</font><font face="Calibri">DR-II Buffer</font><font face="宋體">，均勻混合。當分離小于</font><font face="Calibri">400 bp</font><font face="宋體">的</font><font face="Calibri">DNA</font><font face="宋體">片段時，應在此溶液中再加入終濃度為</font><font face="Calibri">20%</font><font face="宋體">的異丙醇。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">將試劑盒中的</font><font face="Calibri">Spin Column</font><font face="宋體">安置于</font><font face="Calibri">Collection Tube</font><font face="宋體">上。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">將上述操作</font><font face="Calibri">7</font><font face="宋體">的溶液轉移至</font><font face="Calibri">Spin Column</font><font face="宋體">中，</font><font face="Calibri">12000 rpm</font><font face="宋體">離心</font><font face="Calibri">1min</font><font face="宋體">，棄濾液。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;"><font face="宋體">注意：</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">如將濾液再加入</font>Spin Column<font face="宋體">中離心一次，可以提高</font><font face="Calibri">DNA</font><font face="宋體">的回收率。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">將</font><font face="Calibri">500 &micro;L</font><font face="宋體">的</font><font face="Calibri">Rinse A</font><font face="宋體">加入</font><font face="Calibri">Spin Column</font><font face="宋體">中，</font><font face="Calibri">12000 rpm</font><font face="宋體">離心</font><font face="Calibri">30s</font><font face="宋體">，棄濾液。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">將</font><font face="Calibri">700 &micro;L</font><font face="宋體">的</font><font face="Calibri">Rinse B</font><font face="宋體">加入</font><font face="Calibri">Spin Column</font><font face="宋體">中，</font><font face="Calibri">12000 rpm</font><font face="宋體">離心</font><font face="Calibri">30s</font><font face="宋體">，棄濾液。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">重復操作步驟</font><font face="Calibri">11</font><font face="宋體">。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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="宋體">將</font><font face="Calibri">Spin Column</font><font face="宋體">安置于新的</font><font face="Calibri">1.5 mL</font><font face="宋體">的離心管上，在</font><font face="Calibri">Spin Column</font><font face="宋體">膜的中央處加入</font><font face="Calibri">25 &micro;L</font><font face="宋體">的滅菌蒸餾水或</font><font face="Calibri">Elution Buffer</font><font face="宋體">，室溫靜置</font><font face="Calibri">1min</font><font face="宋體">。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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>