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Team:Jilin China/RESULT
From 2014.igem.org
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<p >Make sure that the synthesis report is consistent to the OD number on the primer label,and check the number of the dispensing tube before the dissolution of oligonucleotides.The volume of water added to make 10umol/L oligonucleotides’ solution is calculated according to the synthetic single report of each primer. Add sterile deionized water and keep it at room temperature for 2min.And reverse the tube to accelerate the progress of solubilization. Finally, collect substances at the bottom of the tube after centrifugation.</p> | <p >Make sure that the synthesis report is consistent to the OD number on the primer label,and check the number of the dispensing tube before the dissolution of oligonucleotides.The volume of water added to make 10umol/L oligonucleotides’ solution is calculated according to the synthetic single report of each primer. Add sterile deionized water and keep it at room temperature for 2min.And reverse the tube to accelerate the progress of solubilization. Finally, collect substances at the bottom of the tube after centrifugation.</p> | ||
<p >Every six primers are combined to one group.Add 4ulbeginning fiprimer, 1ulintermediate primer and 4ul end primerto a new EP tube, then add sterile deionized water to 20ul, mix them up as spare.</p> | <p >Every six primers are combined to one group.Add 4ulbeginning fiprimer, 1ulintermediate primer and 4ul end primerto a new EP tube, then add sterile deionized water to 20ul, mix them up as spare.</p> | ||
| - | <table | + | <table border="1" cellspacing="0" cellpadding="0" width="612"> |
| - | <tr > | + | <tr> |
| - | <td width="67" | + | <td width="67"><p align="center"><strong>Group</strong></p></td> |
| - | <td width="103" | + | <td width="103"><p align="center"><strong>Number of primer</strong></p></td> |
| - | <td width="65" | + | <td width="65"><p align="center"><strong>Uptake</strong></p></td> |
| - | <td width="85" | + | <td width="85"><p align="center"><strong>The volume of sterile deionized water</strong></p></td> |
| - | <td width=" | + | <td width="75"><p align="center"><strong>The total volume</strong></p></td> |
| - | <td width="108" valign="top" ><p > | + | <td width="108" valign="top"><p align="center"><strong>Each PCR (50ul system) uptake</strong></p></td> |
| - | <td width="108" | + | <td width="108"><p align="center"><strong>Final concentration</strong></p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="6"><p align="center">A1</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A101</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" | + | <td width="75" rowspan="6"><p align="center"><a name="_GoBack"></a>20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="6"><p align="center">5ul</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A102</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A103</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A104</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A105</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A106</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="6"><p align="center">A2</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A107</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" | + | <td width="75" rowspan="6"><p align="center">20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="6"><p align="center">5ul </p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A108</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A109</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A110</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A111</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A112</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="6"><p align="center">A3</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A113</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" | + | <td width="75" rowspan="6"><p align="center">20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="6"><p align="center">5ul </p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A114</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A201</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A202</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A203</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A204</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="6"><p align="center">A4</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A205</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" | + | <td width="75" rowspan="6"><p align="center">20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="6"><p align="center">5ul </p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A206</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A207</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A208</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A209</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A210</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="4"><p align="center">A5</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A211</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="4"><p align="center">10</p></td> |
| - | <td width=" | + | <td width="75" rowspan="4"><p align="center">20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="4"><p align="center">5ul </p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A212</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A213</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A214</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<h3 >3、DA-PCR </h3> | <h3 >3、DA-PCR </h3> | ||
| - | <p > | + | <p >Every six primers which are single-stranded oligonucleotidesare chemically synthesized were combined to one group. Then they were going to be made as longer double-stranded fragments of intermediate (Block) by DA-PCR </p> |
<p > Mixed primer solutions 5μl </p> | <p > Mixed primer solutions 5μl </p> | ||
<p > Pfu DNA Polymerase 2.5U 0.5μl </p> | <p > Pfu DNA Polymerase 2.5U 0.5μl </p> | ||
| Line 774: | Line 774: | ||
<p > </p> | <p > </p> | ||
<h3 >4、OE-PCR </h3> | <h3 >4、OE-PCR </h3> | ||
| - | <p > | + | <p >Take the 5 double strand intermediate fragment (Block) spliced by DA-PCR as templates, blocks were further connected by OR-PCR, OE-PCR in a reaction system consisting of: </p> |
<p > Block1 1μl </p> | <p > Block1 1μl </p> | ||
<p > Block2 1μl </p> | <p > Block2 1μl </p> | ||
| Line 822: | Line 822: | ||
| - | <h3 > | + | <h3 >5、Constructing and sequencing of subcoloning vector</h3> |
| - | <p > | + | <p >After double enzyme digestion reaction at 37℃ for 3h by EcoRⅠand PstⅠ, MlrA gene and pSB1C3 vector linked together at 16℃ overnight. And then transformed to E.coli JM109 competent cell, and select the desirable colony by using “blue-white selection” method.</p> |
| - | <p > | + | <p >Constitute of EcoRⅠand PstⅠdouble enzyme digestion reaction system：： </p> |
<p > Gene or plasmid 10μl </p> | <p > Gene or plasmid 10μl </p> | ||
<p > EcoRⅠ 0.4μl </p> | <p > EcoRⅠ 0.4μl </p> | ||
| Line 830: | Line 830: | ||
<p > 10×NEBuffer 3.1 2μl </p> | <p > 10×NEBuffer 3.1 2μl </p> | ||
<p > 灭菌超纯水 补齐至 20μl </p> | <p > 灭菌超纯水 补齐至 20μl </p> | ||
| - | <p > | + | <p >Constitute of linking reaction system： </p> |
<p > mlrA Gene 10μl </p> | <p > mlrA Gene 10μl </p> | ||
<p > pSB1C3 3μl </p> | <p > pSB1C3 3μl </p> | ||
Revision as of 01:40, 18 October 2014
Welcome!
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2、Mixing of primersOligonucleotides synthesized in powder form need to be centrifuged for 1 min at 10000rpm to assemble powders to the bottom of the EP tube.And be careful to open the cap. Make sure that the synthesis report is consistent to the OD number on the primer label,and check the number of the dispensing tube before the dissolution of oligonucleotides.The volume of water added to make 10umol/L oligonucleotides’ solution is calculated according to the synthetic single report of each primer. Add sterile deionized water and keep it at room temperature for 2min.And reverse the tube to accelerate the progress of solubilization. Finally, collect substances at the bottom of the tube after centrifugation. Every six primers are combined to one group.Add 4ulbeginning fiprimer, 1ulintermediate primer and 4ul end primerto a new EP tube, then add sterile deionized water to 20ul, mix them up as spare.
3、DA-PCREvery six primers which are single-stranded oligonucleotidesare chemically synthesized were combined to one group. Then they were going to be made as longer double-stranded fragments of intermediate (Block) by DA-PCR Mixed primer solutions 5μl Pfu DNA Polymerase 2.5U 0.5μl dNTP 4μl 10×Pfu buffer（Mg2+） 5μl 灭菌超纯水 补齐至 50μl DA-PCR的反应程序为： 94℃ 2min 94℃ 30S 47℃ 30S 25个循环 72℃ 1min 72℃ 10min 4℃ 保存 End DA-PCR拼接反应产物检测采用2％的琼脂糖凝胶电泳，其中 DA-PCR产物 5μl 10×Loading Buffer 0.6μl 混合均匀后点样 100bp Marker 5μl 直接点样 75V电泳1h。
图1 DA-PCR拼接结果 结果说明： 1、A1、A2、A3、A4都能得到6条单链寡核苷酸拼接到一起的中间产物（Block1-4），但是A3和A4组还可看到4条单链寡核苷酸拼接到一起的副产物，需胶回收后再进一步拼全基因。 2、A5组可得到4条单链寡核苷酸拼接到一起的中间产物（Block5）。
4、OE-PCRTake the 5 double strand intermediate fragment (Block) spliced by DA-PCR as templates, blocks were further connected by OR-PCR, OE-PCR in a reaction system consisting of: Block1 1μl Block2 1μl Block3 1μl Block4 1μl Block5 1μl A1 1μl A4 1μl Pfu DNA Polymerase 2.5U 0.5μl dNTP 4μl 10×Pfu buffer（Mg2+） 5μl
OE-PCR反应程序为： 94℃ 2min
47℃ 30S 20个循环 72℃ 2min 72℃ 10min
End OE-PCR拼接得到的全长基因采用1％的琼脂糖凝胶电泳进行检测，其中
10×Loading Buffer 0.6μl 混合均匀后点样
80V电泳1h。
图2 OE-PCR拼接结果 结果说明： 1、L1、L2、L3都能得到5条中间产物拼接到一起的mlrA全基因。 2、100bp处的宽条带是由于扩增引物A1和A4加入量过多所导致。 5、Constructing and sequencing of subcoloning vectorAfter double enzyme digestion reaction at 37℃ for 3h by EcoRⅠand PstⅠ, MlrA gene and pSB1C3 vector linked together at 16℃ overnight. And then transformed to E.coli JM109 competent cell, and select the desirable colony by using “blue-white selection” method. Constitute of EcoRⅠand PstⅠdouble enzyme digestion reaction system：： Gene or plasmid 10μl EcoRⅠ 0.4μl PstⅠ 0.2μl 10×NEBuffer 3.1 2μl 灭菌超纯水 补齐至 20μl Constitute of linking reaction system： mlrA Gene 10μl pSB1C3 3μl T4 ligase 0.5μl 10×T4 ligase Buffer 2μl 灭菌超纯水 补齐至 20μl E.coli JM109感受态细胞制备流程： (1) E.coli JM109菌株37℃过夜培养以复苏菌种，取过夜培养的菌液600uL加入到30 mL LB培养基中，37℃，200 r/min振荡培养1-2 h，至OD600约为0.3-0.4左右。 (2) 无菌条件下，将30mL菌液转移至离心管中，冰上放置10 min，使培养物冷却至0℃。 (3) 在预冷4℃的离心机上以4000r/min离心10min，弃去上清，加入30mL预冷的0.1mol/L CaCl2溶液重悬沉淀，冰浴上放置10 min。 (4) 以4000r/min在4℃离心10min，弃去上清，每30mL初始培养物用1.0mL预冷的0.1mol/LCaCl2溶液重悬细胞沉淀，200μL/管分装。 热激转化过程： (1) 取新鲜制备或者从-80℃冰箱中取出感受态细胞JM109悬液，冰浴解冻。 (2) 加入构建好的重组克隆载体质粒pSB1C3-A(含量不超过50ng,体积不超过10μl)，轻轻摇匀，冰上放置30分钟后。 (3) 将该EP管放入预加温至42℃的水浴中，放置90s，快速转移到冰浴中，使细胞冷却1-2 min。 (4) 向EP管加入800 μL LB培养基，转移至37℃摇床上，150r/min，温育45 min以复苏菌株。 平板涂布及培养过程： (1) 将37℃培养后的菌体低速离心（5000rpm、5min）浓缩为200μl，涂布到含有氯霉素的LB平板上，设阳性对照。 (2) 将平板置于室温下至液体被完全吸收，倒置平皿，37℃过夜培养； 实验连接组：连接产物，氯霉素板 阳性对照组: 氯霉素板，以pSB1C3质粒代替DNA溶液, 其它操作与上面相同。此组正常情况下在含抗生素的LB平板上应产生大量菌落 阴性对照组：氯霉素板，为连接时用水代替酶，其它与实验组相同。 空菌组：感受态细胞，无氯霉素板 序列测定 在pSB1C3载体上，将EcoR I和Pst I之间的45bp片段使用mlrA的1024bp的片段替换，将得到重组的pSB1C3-A质粒（图3）。
图3 重组载体pSB1C3-A 挑取白色菌落，经液体培养后，提取的质粒作为模版，以VF2及VR作为扩增引物进行PCR鉴定。有外源基因mlrA插入的重组载体，扩增得到的片段长度为1293bp，而无外源插入的pSB1C3空载体，其扩增产物为314bp。结果（图4）表明，在1300bp处，可见到清晰、特异性的扩增产物条带，所以筛选得到的白斑菌落是含有外源基因的重组载体。
图4 重组载体pSB1C3-A的PCR鉴定 结果说明： 1、L1、L2都能扩增得到1300bp的产物，鉴定的重组载体含有外源基因mlrA 2、M是100bp Ladder Marker 将经过PCR鉴定的含有重组载体pSB1C3-A的阳性克隆送到吉林库美生物科技限公司进行测序。结果表明（图5），合成的mlrA基因序列与设计序列一致。
图5 测序结果与设计序列比对
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灭菌超纯水 补齐至 50μl 
94℃ 30S 
4℃ 保存 
OE-PCR产物 5μl 
100bp Marker 5μl 直接点样 
6、引物合成
|
引物编号 |
序列，(5' to 3') |
碱基数（bp） |
合成总量(OD) |
分装管数 |
纯化方式 |
A101 |
CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCT |
60 |
1 |
1 |
PAGE |
A102 |
CTCGTAAAGCATGAGCAGCTAAAGCAATTAAAATAGCTAAAACATAAAAACATAATA |
57 |
1 |
1 |
PAGE |
A103 |
AGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTT |
60 |
1 |
1 |
PAGE |
A104 |
AGCAGTAATAATATTTAAATGAGCATGAGTTTCTTGTAACATTTTAAACATTGGATCTAA |
60 |
1 |
1 |
PAGE |
A105 |
CTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTT |
60 |
1 |
1 |
PAGE |
A106 |
CAGCAAACATTGGAGCAGCTGGAAATAATAATAAAGTATAAGCTCCTGGATATTCA |
56 |
1 |
1 |
PAGE |
A107 |
GCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTT |
60 |
1 |
1 |
PAGE |
A108 |
TCGCCATGGAGCACATCGTGATAATAATTCTCGAAATCCAGCTTGTCCATATCCAA |
56 |
1 |
1 |
PAGE |
A109 |
ACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGT |
60 |
1 |
1 |
PAGE |
A110 |
CATAATTCCAGTTAAAGCAAAAAAAGCTAAAAAACAAACAGCAATAACAGTAACTCCTTG |
60 |
1 |
1 |
PAGE |
A111 |
TTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTT |
60 |
1 |
1 |
PAGE |
A112 |
CAACTGGATCTGATCCATATCGTAAAAAAGTTCGATCTAAAGTTCCTGGTGGAGCATATA |
60 |
1 |
1 |
PAGE |
A113 |
CGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCA |
60 |
1 |
1 |
PAGE |
A114 |
ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAGCAGCT |
44 |
1 |
1 |
PAGE |
A201 |
ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCC |
60 |
1 |
1 |
PAGE |
A202 |
CACCACATAATTCCTAAAATAACAGCAGCAGTTAATGGATCAAATTTTTTTAATAATTGT |
60 |
1 |
1 |
PAGE |
A203 |
TGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATT |
60 |
1 |
1 |
PAGE |
A204 |
AACTGACCAAGCAGCTCCTGGAGCTCCTGAAAATAAAGTTGGTAAATCTCGTGGT |
55 |
1 |
1 |
PAGE |
A205 |
AGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTC |
60 |
1 |
1 |
PAGE |
A206 |
CCTAATTTATTACAAACAAAAACAGCAATAATAGTTGAAGCAATAAATCCTGGAGTAATA |
60 |
1 |
1 |
PAGE |
A207 |
TGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGC |
57 |
1 |
1 |
PAGE |
A208 |
CATTCAGCAGTAACATTAACTCCTAATTCATTATGAATAGCATGAGTTAAAACTCCTCCC |
60 |
1 |
1 |
PAGE |
A209 |
AGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGA |
55 |
1 |
1 |
PAGE |
A210 |
ACTAATCCAATAGCAACAGCAAATTCAATTAAATCCCATGGTCGCCATCCTAATCCAGCA |
60 |
1 |
1 |
PAGE |
A211 |
ATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTC |
57 |
1 |
1 |
PAGE |
A212 |
GTGGAACATTTCCCCAAGCTAATCGAGCATTATCTGGTGAAGCAGCTCCTAATGATCGT |
59 |
1 |
1 |
PAGE |
A213 |
TTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGC |
59 |
1 |
1 |
PAGE |
A214 |
AAGACGTCCTATTAAGCATTAGCTCCTGATTTATCTCCAACTC |
43 |
1 |
1 |
PAGE |
A1 |
CCGGAATTCATGCGAGAATTTGTTCG |
26 |
1 |
1 |
PAGE |
A2 |
ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAG |
39 |
1 |
1 |
PAGE |
A3 |
ATTAGAAGAATTAGGATGGCGAGGATTTGC |
30 |
1 |
1 |
PAGE |
A4 |
AAGACGTCCTATTAAGCATTAGCTCCTG |
28 |
1 |
1 |
PAGE |
A5 |
AACTGCAGCTATTAAGCATTAGCTCCTG |
28 |
|
|
|
