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| − | <div class=" divPanel notop page- content"> | + | <div class=" blog- header" > |
| | + | <h1 class="blog-title">Flight iGEM</h1> |
| | + | <p class="lead blog-description">The official example template of creating an iGEM page with Fligh iGEM.</p> |
| | + | </div> |
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| − | <div class=" breadcrumbs" > | + | |
| − | <a href="index.html">Home</a> / <span>Simple</span>
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| − | | + | <div class=" col- sm- 8 blog- main"> |
| − | <div class="span8" id="divMain">
| + | <volist name="subsections" id="sub"> |
| − | <h1>igem-2015实验总纲</h1>
| + | <div class="blog-post"> |
| − | <p><p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">材料：</span></span></p>
| + | <h2 class="blog-post-title">Sample blog post</h2> |
| − | | + | <p class=" blog- post- meta"> January 1, 2014 by < a href=" http:// getbootstrap. com/ examples/ blog/ #"> Mark</ a></p> |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">菌株：保加利亚乳杆菌、鼠伤寒沙门氏菌、哈氏弧菌BB152、MC1061（待买）</span></span></p> | + | </div><!-- /.blog-post --> |
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">质粒：PNZ8148、PHY300PLK、PBBR1MCS-5、PNZ9530（是否购买看实验结果）</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">一：</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">基因克隆</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">A：多一个碱基→初步采用SOE-PCR解决</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">B：正常</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">C：正常（中间含一段碱基序列，但未造成移码）</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">D：多一个碱基→初步采用SOE-PCR解决</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">R：正常</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">K：未克隆出</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">二：</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif"><strong>质粒构建：</strong></span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1）ABCDRK+ PNZ 8148（问题：连好T-5载体的质粒酶切切不开！？）</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2）DRK+PBBR3MCS-5（初步采用PCR）</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3）PET28b（含蓝光色素蛋白），启动子替换（酶切）→+PHY300PLK</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4）PNZ 8148+蓝光色素蛋白→证明启动子好用</span></span></p>
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| − | <p>5）plsr+蓝光色素蛋白+ PNZ 8148→证明启动子好用</p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif"><strong>大肠杆菌（</strong><strong>MC1061</strong><strong>）感受态制备：</strong></span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">主要试剂</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（1）0.1mol/L CaCl2溶液</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（2）LB液体培养基</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（3）30%甘油：30mL甘油溶于100mL蒸馏水，高压灭菌。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">主要设备</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（1）超净工作台</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（2）冷冻离心机</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（3）恒温摇床</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（4）－70℃冰箱</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（5）10mL移液管</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（6）吸耳球</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（7）1mL、200μL移液枪（配套枪头）</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（8）50mL 离心管</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（9）1.5mL离心管</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">实验材料：大肠杆菌</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">实验步骤</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（一）受体菌的培养</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（1）从LB平板上挑取新活化的E. coli DH5α单菌落，接种于3～5mL LB液体培养基中，37℃下振荡培养过夜（12h左右）。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（2）将该菌种悬液以1:100的比例接种，取250μL菌液转接到25mL LB液体培养基中，37℃振荡培养2～3h至OD600＝0.5左右。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（二）感受态细胞的制备（注意：以下操作在超净工作台完成。）</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（1）将菌液转入50mL离心管中，冰上放置10min。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（2）在4℃下，4000r/min离心10min。弃去上清，将管倒置1min以便培养液流尽。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（3）用冰上预冷的0.1mol/L的CaCl2 溶液10mL轻轻悬浮细胞，冰上放置30min。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（4）0～4℃ 4000r/min离心10min，弃去上清，加入2mL预冷的0.1mol/L的CaCl2 溶液，轻轻悬浮细胞，冰上放置（务必冰上放置）。（注意：以上操作完成了新鲜感受态细胞的制备）</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（三）感受态细胞的分装与冻存</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（1）在2mL制备好的感受态细胞中加入2mL30%甘油（即1:1体积，甘油终浓度15%）。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（2）将此感受态细胞分装成每份200μL （1.5mL dorf管），液氮速冻，快速转入-70℃冰箱保存。（如果没有液氮，可以将分装的感受态细胞直接转入-70℃冰箱保存。）</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">注意事项</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">⑴、细胞的生长状态和密度</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">最好从-70℃或-20℃甘油保存的菌种中直接转接用于制备感受态细胞的菌液。不要用已 经过多次转接，及贮存在4℃的培养菌液。细胞生长密度以每毫升培养液中的细胞数在5×107个左右为佳。即应用对数期或对数生长前期的细菌，可通过测定培养液的OD600控制。对TG1菌株，OD600为0．5时，细胞密度在5×107个/ml左右。（应注意OD600值与细胞数之间的关系随菌株的不同而不同）。密度过高或不足均会使转化率下降。 此外，受体细胞一般应是限制-修饰系统缺陷的突变株，即不含限制性内切酶和甲基化酶的突变株。并且受体细胞还应与所转化的载体性质相匹配。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">⑵、试剂的质量</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">所用的CaCl2等试剂均需是最高纯度的，并用最纯净的水配制，最好分装保存于4℃。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">⑶、防止杂菌和杂DNA的污染</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">整个操作过程均应在无菌条件下进行，所用器皿，如离心管，移液枪头等最好是新的，并经高压灭菌处理。所有的试剂都要灭菌，且注意防止被其它试剂、DNA酶或杂DNA所污染，否则均会影响转化效率或杂DNA的转入。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">⑷、整个操作均需在冰上进行，不能离开冰浴，否则细胞转化率将会降低。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">三：</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">AI-2的化学检测：</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">标品：抗坏血酸</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">标准曲线：抗坏血酸标准曲线</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">试剂：</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1、10 mM 1，10-phenanthroline/3.32 mM Fe（III） ：1，10-phenanthroline（0.198 g）+去离子蒸馏水（50 mL），用1 M HCl调pH 2。</span></span></p>
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| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2、<a name="OLE_LINK2"></a><a name="OLE_LINK1">硫酸铁铵</a> （0.16 g）加到去离子蒸馏水（100 mL）中。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">步骤：</span></span></p> | + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">标准：0.5 mM ascorbic acid</span></span></p> | + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">对于Fe（III）离子减少的检测 ，1 mL 样品（colony rinse in SNSS or cell free supernatant from a bacterial culture） +1 mL Fe（III）-1，10- phenanthroline 反应1 min ，用水稀释到5 mL，过0.2 μm滤膜，510nm测吸光度。</span></span></p> | + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">Negative control：培养基without ascorbic acid （20 μM）</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">positive control：培养基with ascorbic acid （20 μM）</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">干扰因素：</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.5Mammonium acetate</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.5M sodium hydroxide and</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.5 M N-（ß-ketocaproyl）-homoserine lactone （3-oxo-C6-AHL）</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">四：</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">保加利亚乳酸菌感受态的制备及电转化</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">实验前准备</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1、 筛选转化子的抗生素浓度的确定</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">分别配制 含 1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0、13.0、14.0 μg/mL 抗生素的 MRS 抗生素平板，涂布生长至对数期的保加利亚乳杆菌，37℃静止培养 36～48 h 观察结果， 根据抗生素平板上乳杆菌的生长情况确定抗生素的浓度。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2、保加利亚乳杆菌菌株在不同培养基中生长曲线的测定</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">将单菌落接入 10 mL MRS 培养液中，37 ℃静置培养过夜后， 以 2%接种量分别接入 100 mL含 2.5%甘氨酸的液体SMRS 培养基中，37 ℃静置培养，每隔 2 h 取样，于波长 600 nm处测 OD 值。得到对数中期的时间（文献中为8h）</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">乳杆菌感受态细胞的制备与电转化</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1、将单菌落接入10 mL MRS 培养液中，37 ℃静置培养过夜</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2、感受态细胞的培养：（以 2%接种量分别接入100 mL 2.5%甘氨酸的液体 SMRS 培养基中 37 ℃静置培养，冷却（将培养物置于冰上 20～30 min），离心洗涤与细胞重悬（6 000 r/min 离心 10 min，收集菌体，用 PB 缓冲液洗涤 3 次后，再用1 mL PB 缓冲液重悬菌体）。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3、感受态细胞与质粒 DNA 混合（吸取 50 μL 新制备的感受态细胞悬液到预冷的无菌EP 管中，加入 1 μL 质量浓度为 1 μg/μL 的质粒，冰浴 5 min， 将此混合液加入预冷的 2 mm 电转化杯中）高压脉冲电击转化（电场强度 12 kV/cm，电阻 200 Ω、电容 25 μF）。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4、转化子复苏（电击完毕后迅速向电转化杯中加入950 μL含有 20 mmol/L（MgCl2、CaCl2）的 SMRS 液体培养基，混匀后倒入无菌 EP 管中，37 ℃静置培养2 h）。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">5、转化子培养与筛选 （取用再生液体培养基稀释后的电击转化细胞液 100 μL，涂布到含相应浓度的抗生素的 MRS 平板上，37 ℃倒置培养 36～48 h 后，计数转化子并计算转化效率）。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">五：</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">表达产物分析：</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">RNA水平：多重PCR</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">蛋白质水平：SDS-PAGE</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">制 胶</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1、试 剂</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1） 丙烯酰胺（电泳级）</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2） 双丙烯酰胺（N，N—甲叉双丙烯酰肢）</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3） Tris 絨</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4） SDS （十二烷基磺酸钠）</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">5） TEMED</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">6） 过硫酸铵</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">7） α-巯基乙醇</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">8） 甘油</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">9） 溴酚蓝</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">10）甘氨酸</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">11）盐酸</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">12） DTT</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2、储存液</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（1） 2M Tris-HCl（pH8.8），100ml</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">称取 24.2g Tris 碱；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加50ml蒸馏水；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">缓慢的加浓盐酸至pH8.8 （约加4ml）；让溶液冷却至室温，pH将会升高；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至100ml</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（2）1M Tris-HCI（pH6.8），100ml</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">称取12.1g的Tris碱；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加50ml蒸馏水；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">缓慢的加浓盐酸至pH6.8（约加8ml）；让溶液冷却至室温，pH将会升高L；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至总量100ml.</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（3）10%（w/v）SDS，100ml 室温保存</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif"> 称取10g的SDS；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至总量为100ml。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（4）50%（v/v）甘油，100ml</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">到取 50ml 100%的甘油；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加人5ml蒸馏水。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（5）1% （w/v）溴酚蓝，10ml</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">称取100mg溴酚蓝；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至10ml，搅拌直到完全溶解；</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">过滤除去聚合的染料。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3、 工作液</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（1） A液：丙烯酰胺储存液，100ml</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">30% （w/v）丙烯酰胺，0.8%（w/v）双丙烯酰胺。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">29.2g丙烯酰胺</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.8g双丙烯酰胺</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（2）B液：4X分离胶缓冲液，100ml</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">75ml 2 mol/L Tris-HCl（pH8.8） 1.5 mol/L</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4ml 10% SDS 0.4%</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">21ml蒸馏水</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">可在4°C存放数月。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（3）C液：4X堆积胶缓冲液，100ml</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">50ml 1 mol/L Tris-HCI （pH6.8） 0.5mol/L</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">4ml 10%SDS 0.4%</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">46ml蒸馏水 可在41：存放数月。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（4） 10%过硫酸铵，5m丨 0.5g过硫酸铵</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">5mJ蒸溜水</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">可在密封的管内，4C存放数月。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（5） 电泳缓冲液，1L</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">3g Tris 碱 25mM</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">14.4g 甘氨酸 192mM</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">IgSDS 0-1%</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">加蒸馏水至1L</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">pH应该在8.3左右。也可以制成10x的储存液，在室 温下长期保存。</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">（6）5X样品缓冲液，10ml</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.6ml 1mol/L 的Tris-HCl（pH6.8） 60mmol/L</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">5ml 50%的甘油 25%</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">2ml 10%的SDS 2%</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.5ml 2-巯基乙醇 14.4mmol/L </span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">1ml 1％溴酚蓝 0.1%</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">0.9ml的蒸馏水</span></span></p>
| + | |
| − | | + | |
| − | <p><span style="font-size:10px"><span style="font-family:arial,helvetica,sans-serif">可在4°C保存数周，或许-20C保存数月</span></span></p>
| + | |
| − | | + | |
| − | <p> </p>
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| − | <div id="divFooter" class="footerArea">
| + | <div class="col-sm-3 col-sm-offset-1 blog-sidebar"> |
| − | | + | <div class=" sidebar- module sidebar-module-inset"> |
| − | <div class="divPanel"> | + | <h4>About</h4> |
| − | | + | <p> Etiam porta < em> sem malesuada magna</ em> mollis euismod. Cras mattis consectetur purus sit amet fermentum. Aenean lacinia bibendum nulla sed consectetur.</ p> |
| − | | + | </div> |
| − | <div class="span3" id="footerArea1">
| + | <div class="sidebar-module"> |
| − |
| + | <h4>Archives</h4> |
| − | <h3>About Company</h3>
| + | <ol class="list-unstyled"> |
| − | | + | < li><a href="http:// getbootstrap. com/ examples/ blog/ #"> Project</ a></ li> |
| − | <p> Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry’s standard dummy text ever since the 1500s.< /p> | + | < li><a href="http:// getbootstrap. com/ examples/ blog/ #"> Team</ a></ li> |
| − |
| + | < li><a href="http:// getbootstrap. com/ examples/ blog/ #"> Safety</ a></ li> |
| − | <p>
| + | < li><a href="http:// getbootstrap. com/ examples/ blog/ #"> Parts</a></li> |
| − | <a href="#" title="Terms of Use">Terms of Use</a><br />
| + | <li><a href="http://getbootstrap.com/examples/blog/#">Human practise</a></li> |
| − | <a href="#" title="Privacy Policy">Privacy Policy</a><br />
| + | </ol> |
| − | <a href="#" title="FAQ">FAQ</a><br />
| + | </div> |
| − | <a href="#" title="Sitemap">Sitemap</a>
| + | <div class="sidebar-module"> |
| − | </p>
| + | <h4>Elsewhere</h4> |
| − | | + | <ol class="list-unstyled"> |
| − | </div>
| + | < li><a href="http:// getbootstrap.com/ examples/blog/#"> GitHub</a></ li> |
| − | <div class="span3" id="footerArea2">
| + | < li><a href=" http://getbootstrap.com/examples/blog/#"> Twitter< /a></ li> |
| − | | + | < li><a href=" http:// getbootstrap.com/ examples/ blog/#"> Facebook</a></ li> |
| − | <h3>Recent Blog Posts</h3>
| + | </ol> |
| − | <p>
| + | </div> |
| − | <a href="http:// www. oswt.co.uk/ " title="">Lorem Ipsum is simply dummy text</ a><br / > | + | </div><!-- /.blog-sidebar --> |
| − | <span style="text-transform:none;">2 hours ago</span>
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <a href="http:// www. oswt.co.uk/ " title="">Duis mollis, est non commodo luctus</ a><br / > | + | |
| − | <span style="text-transform:none;">5 hours ago</span>
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <a href="http:// www. oswt.co.uk/ " title="">Maecenas sed diam eget risus varius</ a><br / > | + | |
| − | <span style="text-transform:none;">19 hours ago</span>
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <a href="http:// www. oswt.co.uk/ " title="">VIEW ALL POSTS</ a> | + | |
| − | </p>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | <div class="span3" id="footerArea3">
| + | |
| − | | + | |
| − | <h3>Sample Footer Content</h3>
| + | |
| − | <p>Lorem Ipsum has been the industry’s standard dummy text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. </p>
| + | |
| − | <p>Duis mollis, est non commodo luctus, nisi erat porttitor ligula, eget lacinia odio sem nec elit.</p>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | <div class="span3" id="footerArea4">
| + | |
| − | | + | |
| − | <h3>Get in Touch</h3>
| + | |
| − |
| + | |
| − | <ul id="contact-info">
| + | |
| − | <li>
| + | |
| − | <i class="general foundicon-phone icon"></i>
| + | |
| − | <span class="field">Phone:</span>
| + | |
| − | <br />
| + | |
| − | (123) 456 7890 / 456 7891
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <i class="general foundicon-mail icon"></i>
| + | |
| − | <span class="field">Email:</span>
| + | |
| − | <br />
| + | |
| − | <a href="mailto:info@yourdomain.com" title="Email">info@yourdomain.com</a>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <i class="general foundicon-home icon" style="margin-bottom:50px"></i>
| + | |
| − | <span class="field">Address:</span>
| + | |
| − | <br />
| + | |
| − | 123 Street<br />
| + | |
| − | 12345 City, State<br />
| + | |
| − | Country
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <br /><br /> | + | |
| − | <div class="row-fluid"> | + | |
| − | <div class="span12">
| + | |
| − | <p class="copyright">
| + | |
| − | Copyright © 2013 Your Company. All Rights Reserved. More Templates <a href="http:// www.cssmoban.com/ " target="_blank" title="模板之家"> 模板之家</a> | + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="social_bookmarks">
| + | |
| − | <a href="#">< i class="social foundicon-facebook"></ i> Facebook< /a> | + | |
| − | <a href=" https:// twitter.com/ oswt"><i class="social foundicon-twitter"></ i> Twitter</ a> | + | |
| − | <a href="#"><i class="social foundicon-pinterest"></i> Pinterest</a>
| + | |
| − | <a href="#"><i class="social foundicon-rss"></i> Rss</a>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | </div> | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <br /><br /><br />
| + | |
| | | | |
| − | <script src="scripts/jquery-1.8.2.min.js" type="text/javascript"></script> | + | </div><!-- /.row --> |
| − | <script src="scripts/bootstrap/js/bootstrap.min.js" type="text/javascript"></script>
| + | |
| − | <script src="scripts/default.js" type="text/javascript"></script>
| + | |
| | | | |
| | + | </div><!-- /.container --> |
| | | | |
| | + | <footer class="blog-footer"> |
| | + | <p>Blog template built for <a href="http://getbootstrap.com/"></a> by <a href="#">Flight iGEM</a>.</p> |
| | + | <p> |
| | + | <a href="http://getbootstrap.com/examples/blog/#">Back to top</a> |
| | + | </p> |
| | + | </footer> |
| | | | |
| | | | |
| | + | <!-- Bootstrap core JavaScript |
| | + | ================================================== --> |
| | + | <!-- Placed at the end of the document so the pages load faster --> |
| | + | <script src="https://ajax.googleapis.com/ajax/libs/jquery/1.11.3/jquery.min.js"></script> |
| | + | <script src="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.5/js/bootstrap.min.js"></script> |
| | + | <!-- IE10 viewport hack for Surface/desktop Windows 8 bug --> |
| | + | <script src="http://v3.bootcss.com/assets/js/ie10-viewport-bug-workaround.js"></script> |
| | + | |
| | | | |
| − | </body> | + | </body></html> |
| − | </html> | + | |