菌落PCR，快速鉴定重组质粒

<font><font><b><span>质粒快速鉴定</span> </b> <b><span><span> </span> </span></b></font></font>

<font><b><span>试剂：</span> </b> </font>

<font><span>Protoplasting buffer: </span></font>

<font><span>30mM Tris-HCl, pH8.0 <span> </span> 0.33ml/1.0M</span></font>

<font><span><span> </span> <span> </span> 5mM EDTA <span> </span> 0.1ml/0.5M</span></font>

<font><span><span> </span> <span> </span> 50mM NaCl <span> </span> 0.1ml/5.0M</span></font>

<font><span><span> </span> <span> </span> 20% Sucrose <span> </span> <span> </span> 5ml/40%</span></font>

<font><span><span> </span> <span> </span> 50</span> <span>µ</span> <span>g/ml RNAseI <span> </span> 50ul/10mg/ml</span></font>

<font><span><span> </span> <span> </span> 50</span> <span>µ</span> <span>g/ml lysozyme<span> </span> 50ul/10mg/ml</span></font>

<font><span>补水至<span>10ml，-20℃分装保存。</span> </span> </font>

<font><span><span> </span> Lysis buffer:</span></font>

<font><line> <span>89mM Tris-HCl, pH8.0</span></line></font>

<font><line> <span>89mM boric acid<span> </span> 2ml of 5×TBE</span></line></font>

<font><span>2.5mM EDTA</span></font>

<font><span>2% SDS<span> </span> <span> </span> 2ml of<span> </span> 10%</span></font>

<font><span>5% sucrose <span> </span> 1.25ml of 40%</span></font>

<font><span>0.04% bromphenol blue<span> </span> 4mg</span> </font>

<font><span>补水至</span> <span><font>10ml</font> </span> <span>，</span> <span><font>-</font> </span> <span>20℃分装保存。</span> </font>

<font><b><span>步骤：</span> </b> </font>

<font><span><font>1</font> </span> <span>．将转化后的菌液铺平板，</span> <span><font>37</font> </span> <span>℃</span> <span>过夜培养。</span> </font>

<font><span><font>2</font> </span> <span>．配制</span> <span><font>0 .6--0 .7%</font> </span> <span>的琼脂糖</span> <span><font>TBE </font> </span> <span>胶。</span> </font>

<font><span><font>3</font> </span> <span>．用连续加样枪在</span> <span><font>96</font> </span> <span>孔板中每孔加入</span> <span><font>10 </font> </span> <span>µ</span> <font><span>l Photo</span> <span>plasting</span> <span> Buffer</span> </font> <span>。</span> </font>

<font><span><font>4</font> </span> <span>．用灭过菌的</span> <span><font>10ul</font> </span> <span>小枪头挑取单克隆白斑至含有</span> <font><span>Photo</span> <span>plasting</span> <span> Buffer </span> </font> <span>的</span> <span><font>96</font> </span> <span>孔板中，振荡混匀。</span> </font>

<font><span><font>5</font> </span> <span>．用连续加样枪将</span> <span><font>Lysis Buffer</font> </span> <span>上样于凝胶中，每孔</span> <span><font>4 </font> </span> <span>µ</span> <span><font>l</font> </span> <span>，用排枪将细胞与</span> <span><font>Protoplasting buffer</font> </span> <span>混合液</span> <span>上样于凝胶中（细胞在</span> <span><font>Protoplasting buffer</font> </span> <span>中不宜超过</span> <span><font>30-40 min</font> </span> <span>），并点上</span> <span><font>Marker</font> </span> <span>。</span> </font>

<font><span><font>6</font> </span> <span>．调节电压为</span> <span><font>20V</font> </span> <span>（小槽）或</span> <span><font>40V</font> </span> <span>（大槽），电泳</span> <span><font>15min</font> </span> <span>，使细胞充分裂解，将电压调高到</span> <span><font>200V</font> </span> <span>，继续电泳</span> <span><font>1hr</font> </span> <span>，照相。</span> </font>

<font><span><font>7</font> </span> <span>．根据胶图粗略鉴定插入片段的大小及小片段率。</span> </font>

<font><b><span><font>2</font> </span> </b> <b><span>．菌落</span> </b> <b><span><font>PCR</font> </span></b></font>

<font><font><span>1．取适量PCR薄壁管，置于冰上，每管先加入17.3ul的灭菌水。</span></font></font>

<font><font><span>2．用灭过菌的10ul小枪头挑取单克隆白斑至灭菌水中，</span> <span>振荡混匀。</span> </font></font>

<font><font><span>3．依次加入：</span></font></font>

<font><font><span><span> </span> 10xbuffer<span> </span> <span> </span> 2.5<span> </span> <span> </span> </span></font></font>

<font><font><span>Mgcl₂ (25mM) <span> </span> <span> </span> <span> </span> 1.8</span></font></font>

<font><font><span>DNTP(2.5mM)<span> </span> <span> </span> <span> </span> <span> </span> 1<span> </span> <span> </span> </span></font></font>

<font><font><span>T3 引物（10pmol）<span> </span> 1<span> </span> <span> </span> </span></font></font>

<font><font><span>T7 引物（10pmol）<span> </span> 1<span> </span> </span></font></font>

<font><font><u><span>Taq酶<span> </span> 0.4<span> </span> </span></u></font></font>

<font><font><span>total<span> </span> <span> </span> 25ul<span> </span> </span></font></font>

<font><font><span>各试剂均加好后，离心机上甩一下，使之沉底，置于<span>PCR仪上</span></span></font></font>

<font><font><span>4．94℃，2min。</span></font></font>

<font><font><span>5．<span> </span> 94℃<span> </span> 4分钟</span></font></font>

<font><font><line> <line> <span>94℃<span> </span> 40秒</span></line></line></font></font>

<font><font><span>53.6℃<span> </span> 40秒<span> </span> 35个循环</span></font></font>

<font><font><line> <span>72℃<span> </span> 4分钟 </span></line></font></font>

<font><font><span>72℃<span> </span> 10分钟</span></font></font>

<font><font><span>4℃<span> </span> 24小时</span></font></font>

<font><font><span>6．待PCR反应进入4℃后，取下PCR薄壁管，取7ulPCR产物加入3ul溴芬兰跑电泳，同时上1Kb DNA ladder。半小时后照相，观察胶图，</span> <span>根据胶图粗略鉴定插入片段的大小及小片段率。</span> </font></font>

<font><font><span>7．将快速鉴定和菌落PCR检测合格的文库送检。</span></font></font>

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