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| | <div class="menu"> | | <div class="menu"> |
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| | <li><a style="margin-top:20px;" class="hide" >PROJECT<span class="caret"></span></a> | | <li><a style="margin-top:20px;" class="hide" >PROJECT<span class="caret"></span></a> |
| | <ul> | | <ul> |
| - | <li><a style="margin-top:20px;" href="https://2014.igem.org/Team:Jilin_China/NOTEBOOK" title="a coded list of spies">NOTEBOOK</a></li> | + | <li><a style="margin-top:20px;" href="https://2014.igem.org/Team:Jilin_China/Outline" title="a coded list of spies">OUTLINE</a></li> |
| - | <li><a href="https://2014.igem.org/Team:Jilin_China/RESULT" title="a horizontal vertical menu">RESULT</a></li> | + | <li><a href="https://2014.igem.org/Team:Jilin_China/RESULT" title="a horizontal vertical menu">CONTRUST</a></li> |
| | + | <li><a href="https://2014.igem.org/Team:Jilin_China/NOTEBOOK" title="a horizontal vertical menu">TEST</a></li> |
| | + | <li><a href="https://2014.igem.org/Team:Jilin_China/Blueprint" title="a horizontal vertical menu">BLUEPRINT</a></li> |
| | </ul> | | </ul> |
| | </li> | | </li> |
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| | <!-- Block of content 1 --> | | <!-- Block of content 1 --> |
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| - | <a href="#menu" style="text-decoration:none;color:#1C140D"> Top </a> </td></tr>
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| | </head> | | </head> |
| | <!--内容--> | | <!--内容--> |
| - | | + | <div id="coop"> |
| - | <div class="observation"><center><h3>mlrA基因合成</h3></center> | + | <center><h2>Synthesis of gene mlrA</h2></center> |
| - | <p >一、实验方案 </p> | + | <h3>The experimental scheme</h3> |
| - | <p >1、目的蛋白氨基酸序列 </p> | + | <h3>1、The amino acid sequence of the protein</h3> |
| | <p >MlrA GenBank: AF411068	SOURCE Sphingomonas sp. ACM-3962 </p> | | <p >MlrA GenBank: AF411068	SOURCE Sphingomonas sp. ACM-3962 </p> |
| | <p >MREFVRQRPLLCFYVLAILIALAAHALRAMSPTPLDPMFKMLQETHAHLNIITAVRSTFEYPGAYTLLLFPAAPMFAALIATGIGYGQAGFRELLSRCAPWRSPVSWRQGVTVIAVCFLAFFALTGIMWVQTYLYAPPGTLDRTFLRYGSDPVAIYVMLAASLLLSPGPLLEELGWRGFALPQLLKKFDPLTAAVILGIMWWAWHLPRDLPTLFSGAPGAAWSVIVKQLVITPGFIASTIIAVFVCNKLGGSMWGGVLTHAIHNELGVNVTAEWAPTVAGLGWRPWDLIEFAVAIGLVLICGRSLGAASPDNARLAWGNVPPKLPGGVGDKSGANA </p> | | <p >MREFVRQRPLLCFYVLAILIALAAHALRAMSPTPLDPMFKMLQETHAHLNIITAVRSTFEYPGAYTLLLFPAAPMFAALIATGIGYGQAGFRELLSRCAPWRSPVSWRQGVTVIAVCFLAFFALTGIMWVQTYLYAPPGTLDRTFLRYGSDPVAIYVMLAASLLLSPGPLLEELGWRGFALPQLLKKFDPLTAAVILGIMWWAWHLPRDLPTLFSGAPGAAWSVIVKQLVITPGFIASTIIAVFVCNKLGGSMWGGVLTHAIHNELGVNVTAEWAPTVAGLGWRPWDLIEFAVAIGLVLICGRSLGAASPDNARLAWGNVPPKLPGGVGDKSGANA </p> |
| - | <h3 >2、密码子选择 </h3> | + | <h3 >2、The selection of codons</h3> |
| - | <p >Lactococcus lactis subsp. cremoris SK11 [gbbct]: 2504 CDS's (696252 codons) </p> | + | <h4>Lactococcus lactis subsp. cremoris SK11 [gbbct]: 2504 CDS's (696252 codons) </h4> |
| - | <p >http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272622 </p> | + | <p ><a href="http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272622">http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272622</a> </p> |
| | | | |
| | <p >fields: [triplet] [frequency: per thousand] ([number]) </p> | | <p >fields: [triplet] [frequency: per thousand] ([number]) </p> |
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| | <p >GUG 9.2( 6376) GCG 8.2( 5708) GAG 13.0( 9070) GGG 8.2( 5732) </p> | | <p >GUG 9.2( 6376) GCG 8.2( 5708) GAG 13.0( 9070) GGG 8.2( 5732) </p> |
| | <p >Coding GC 36.75% 1st letter GC 48.61% 2nd letter GC 34.45% 3rd letter GC 27.20% </p> | | <p >Coding GC 36.75% 1st letter GC 48.61% 2nd letter GC 34.45% 3rd letter GC 27.20% </p> |
| - | <p >密码子密码子密码子 </p> | + | <h4>The codon table used</h4> |
| - | <p > </p>
| + | |
| - | <p >使用密码子表 </p>
| + | |
| | <p >* TAA </p> | | <p >* TAA </p> |
| | <p >A GCT </p> | | <p >A GCT </p> |
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| | <p >W TGG </p> | | <p >W TGG </p> |
| | <p >Y TAT </p> | | <p >Y TAT </p> |
| - | <p > </p>
| + | <h3>3、The inverse translation sequences and the selection of restriction enzymes</h3> |
| - | <h3 >3、逆翻译序列及限制性内切酶选择 </h3> | + | |
| | <p >EcoR I </p> | | <p >EcoR I </p> |
| | <p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGCTGCAGTT </p> | | <p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGCTGCAGTT </p> |
| - | | + | <h3 >4、The sequence analysis</h3> |
| - | | + | |
| - | <h3 >4、序列分析 </h3> | + | |
| | <p >_Base Count : 1008 bp (270 A, 377 T, 158 C, 203 G)<br> | | <p >_Base Count : 1008 bp (270 A, 377 T, 158 C, 203 G)<br> |
| |  _Composition : 36% GC, 64% AT<br> | |  _Composition : 36% GC, 64% AT<br> |
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| | <p >NlaIV (821) </p> | | <p >NlaIV (821) </p> |
| | <p >Hpy166II (901) </p> | | <p >Hpy166II (901) </p> |
| - | <h3 >5、序列拆分 </h3> | + | <h3 >5、Sequence Split</h3> |
| | <p >The maximum allowable assembly oligo length is equal to the target assembly oligo length (60). This may cause some weird behavior, especially in terms of overlap melting temperature. </p> | | <p >The maximum allowable assembly oligo length is equal to the target assembly oligo length (60). This may cause some weird behavior, especially in terms of overlap melting temperature. </p> |
| | <p > </p> | | <p > </p> |
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| |                                     TGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCAC            TGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAA                 ATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCC            CCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTAC            ACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCA            TGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTG               CTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA<br> | |                                     TGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCAC            TGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAA                 ATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCC            CCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTAC            ACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCA            TGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTG               CTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA<br> |
| | TAATCTTCTTAATCCTACCGCTCCTAAACGAAATGGTGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCACCCGAACCGTAAATGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAATAACAATTTGTTAATCAATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCCTCCTAGTTACACCCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTACCCGAGGTTGACAACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCAAAATTAAACACCTGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTGGTTTTAATGGTCCTCCTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA </p> | | TAATCTTCTTAATCCTACCGCTCCTAAACGAAATGGTGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCACCCGAACCGTAAATGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAATAACAATTTGTTAATCAATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCCTCCTAGTTACACCCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTACCCGAGGTTGACAACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCAAAATTAAACACCTGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTGGTTTTAATGGTCCTCCTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA </p> |
| - | <h2 >二、实验操作及结果分析 </h2> | + | <h3>The experimental procedure and analysis</h3> |
| - | <h3 >1、引物溶解 </h3> | + | <h3 >1、Dissolving of primers</h3> |
| - | <p >根据合成引物的分子量，计算需要添加的灭菌去离子水的量，将合成的引物稀释成10um/L备用。 </p> | + | <p >Sterile deionized water whose volume is determined by primer’s molecular weight is used to dilute the solution of the primer to 10um/L. And it is prepared for the following experiment. </p> |
| | <table align="center" > | | <table align="center" > |
| | <tr > | | <tr > |
| - | <td width="47" valign="center" ><p >引物编号 </p></td> | + | <td width="47" valign="center" ><p >Num</p></td> |
| | <td width="48" valign="center" ><p >Tm </p></td> | | <td width="48" valign="center" ><p >Tm </p></td> |
| | <td width="85" valign="center" ><p >MW(g/mole) </p></td> | | <td width="85" valign="center" ><p >MW(g/mole) </p></td> |
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| | </tr> | | </tr> |
| | </table> | | </table> |
| - | <p > </p>
| + | <h3 id="coop">2、Mixing of primers</h3> |
| - | <h3 >2、引物混合 </h3> | + | <p id="coop">Oligonucleotides 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. </p> |
| - | <p >合成的寡核苷酸呈粉末状，使用前需要10000rpm离心1min，使寡核苷酸干粉集中在管底，使用时小心开启瓶盖。 </p> | + | <p id="coop">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 >溶解寡核苷酸前核对合成报告单与引物标签上的OD数是否一致，并核对分装管数。根据每个引物的合成报告单计算出配制10umol/L浓度的寡核苷酸需要加入水的体积，加入去离子无菌水，室温放置2min，反复振荡助溶，再离心将溶液收集到管底。 </p> | + | <p id="coop">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 >每6条引物为一组，首末位引物取4ul，中间引物取1ul，加入新的EP管中，再补充灭菌去离子水至20ul，混匀备用。 </p> | + | <table align="center" border="1" cellspacing="0" cellpadding="0" width="612"> |
| - | <table align="center" > | + | <tr> |
| - | <tr > | + | <td width="67"><p align="center"><strong>Group</strong></p></td> |
| - | <td width="67" valign="center" ><p >组别 </p></td> | + | <td width="103"><p align="center"><strong>Number of primer</strong></p></td> |
| - | <td width="103" valign="center" ><p >引物编号 </p></td> | + | <td width="65"><p align="center"><strong>Uptake</strong></p></td> |
| - | <td width="65" valign="center" ><p >吸取量 </p></td> | + | <td width="85"><p align="center"><strong>The volume of sterile deionized water</strong></p></td> |
| - | <td width="85" valign="center" ><p >灭菌去离子水添加量 </p></td> | + | <td width="75"><p align="center"><strong>The total volume</strong></p></td> |
| - | <td width="74" valign="center" ><p >总体积 </p></td> | + | <td width="108" valign="top"><p align="center"><strong>Each PCR (50ul system) uptake</strong></p></td> |
| - | <td width="108" valign="top" ><p >每PCR（50ul体系）吸取量 </p></td> | + | <td width="108"><p align="center"><strong>Final concentration</strong></p></td> |
| - | <td width="108" valign="center" ><p >终浓度 </p></td> | + | |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="67 " valign="center" rowspan="6" ><p >A1 </p></td> | + | <td width="67" rowspan="6"><p align="center">A1</p></td> |
| - | <td width="103" valign="center" ><p >A101 </p></td> | + | <td width="103"><p align="left">A101</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 " valign="center" rowspan="6" ><p >8 </p></td> | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width="74" valign="center" rowspan="6" ><p >20 </p></td> | + | <td width="75" rowspan="6"><p align="center"><a name="_GoBack"></a>20</p></td> |
| - | <td width="108 " valign="center" rowspan="6" ><p >5ul </p></td> | + | <td width="108" rowspan="6"><p align="center">5ul</p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A102 </p></td> | + | <td width="103"><p align="left">A102</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="center" ><p >50nM </p></td> | + | <td width="108"><p align="center">50nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A103 </p></td> | + | <td width="103"><p align="left">A103</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A104 </p></td> | + | <td width="103"><p align="left">A104</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A105 </p></td> | + | <td width="103"><p align="left">A105</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A106 </p></td> | + | <td width="103"><p align="left">A106</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="67 " valign="center" rowspan="6" ><p >A2 </p></td> | + | <td width="67" rowspan="6"><p align="center">A2</p></td> |
| - | <td width="103" valign="center" ><p >A107 </p></td> | + | <td width="103"><p align="left">A107</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 " valign="center" rowspan="6" ><p >8 </p></td> | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" 74" valign="center" rowspan="6" ><p >20 </p></td> | + | <td width=" 75" rowspan="6"><p align="center">20</p></td> |
| - | <td width="108 " valign="center" rowspan="6" ><p >5ul </p></td> | + | <td width="108" rowspan="6"><p align="center">5ul </p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A108 </p></td> | + | <td width="103"><p align="left">A108</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="center" ><p >50nM </p></td> | + | <td width="108"><p align="center">50nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A109 </p></td> | + | <td width="103"><p align="left">A109</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A110 </p></td> | + | <td width="103"><p align="left">A110</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A111 </p></td> | + | <td width="103"><p align="left">A111</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A112 </p></td> | + | <td width="103"><p align="left">A112</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="67 " valign="center" rowspan="6" ><p >A3 </p></td> | + | <td width="67" rowspan="6"><p align="center">A3</p></td> |
| - | <td width="103" valign="center" ><p >A113 </p></td> | + | <td width="103"><p align="left">A113</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 " valign="center" rowspan="6" ><p >8 </p></td> | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" 74" valign="center" rowspan="6" ><p >20 </p></td> | + | <td width=" 75" rowspan="6"><p align="center">20</p></td> |
| - | <td width="108 " valign="center" rowspan="6" ><p >5ul </p></td> | + | <td width="108" rowspan="6"><p align="center">5ul </p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A114 </p></td> | + | <td width="103"><p align="left">A114</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="center" ><p >50nM </p></td> | + | <td width="108"><p align="center">50nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A201 </p></td> | + | <td width="103"><p align="left">A201</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A202 </p></td> | + | <td width="103"><p align="left">A202</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A203 </p></td> | + | <td width="103"><p align="left">A203</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A204 </p></td> | + | <td width="103"><p align="left">A204</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="67 " valign="center" rowspan="6" ><p >A4 </p></td> | + | <td width="67" rowspan="6"><p align="center">A4</p></td> |
| - | <td width="103" valign="center" ><p >A205 </p></td> | + | <td width="103"><p align="left">A205</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 " valign="center" rowspan="6" ><p >8 </p></td> | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" 74" valign="center" rowspan="6" ><p >20 </p></td> | + | <td width=" 75" rowspan="6"><p align="center">20</p></td> |
| - | <td width="108 " valign="center" rowspan="6" ><p >5ul </p></td> | + | <td width="108" rowspan="6"><p align="center">5ul </p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A206 </p></td> | + | <td width="103"><p align="left">A206</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="center" ><p >50nM </p></td> | + | <td width="108"><p align="center">50nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A207 </p></td> | + | <td width="103"><p align="left">A207</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A208 </p></td> | + | <td width="103"><p align="left">A208</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A209 </p></td> | + | <td width="103"><p align="left">A209</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A210 </p></td> | + | <td width="103"><p align="left">A210</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="67 " valign="center" rowspan="4" ><p >A5 </p></td> | + | <td width="67" rowspan="4"><p align="center">A5</p></td> |
| - | <td width="103" valign="center" ><p >A211 </p></td> | + | <td width="103"><p align="left">A211</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 " valign="center" rowspan="4" ><p >10 </p></td> | + | <td width="85" rowspan="4"><p align="center">10</p></td> |
| - | <td width=" 74" valign="center" rowspan="4" ><p >20 </p></td> | + | <td width=" 75" rowspan="4"><p align="center">20</p></td> |
| - | <td width="108 " valign="center" rowspan="4" ><p >5ul </p></td> | + | <td width="108" rowspan="4"><p align="center">5ul </p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A212 </p></td> | + | <td width="103"><p align="left">A212</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="center" ><p >50nM </p></td> | + | <td width="108"><p align="center">50nM</p></td> |
| | </tr> | | </tr> |
| - | <tr > | + | <tr> |
| - | <td width="103" valign="center" ><p >A213 </p></td> | + | <td width="103"><p align="left">A213</p></td> |
| - | <td width="65" valign="center" ><p >1 </p></td> | + | <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" valign="center" ><p >A214 </p></td> | + | <td width="103"><p align="left">A214</p></td> |
| - | <td width="65" valign="center" ><p >4 </p></td> | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" valign="center" ><p >200nM </p></td> | + | <td width="108"><p align="center">200nM</p></td> |
| | </tr> | | </tr> |
| | </table> | | </table> |
| | | | |
| | | | |
| - | <h3 >3、DA-PCR </h3> | + | <h3 id="coop">3、DA-PCR </h3> |
| - | <p >化学合成的单链寡核苷酸，每6条分为一组，通过DA-PCR拼接成为较长的双链中间片段（Block）。DA-PCR的反应体系组成为： </p> | + | <p id="coop">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 id="coop">Procedure of DA-PCR： <br> |
| - | <p > Pfu DNA Polymerase 2.5U 0.5μl </p> | + | Mixed primer solutions 5μl<br> |
| - | <p > dNTP 4μl </p> | + | Pfu DNA Polymerase 2.5U 0.5μl <br> |
| - | <p > 10×Pfu buffer（Mg2+） 5μl </p> | + | dNTP 4μl<br> |
| - | <p > 灭菌超纯水 补齐至 50μl </p> | + | 10×Pfu buffer（Mg2+） 5μl<br> |
| - | <p >DA-PCR的反应程序为： </p> | + | ddH20add to 50μl<br> |
| | + | Procedure of DA-PCR： <br> |
| | + | 94℃ 2min<br> |
| | + | 94℃ 30S<br> |
| | + | 50℃ 30S 25 cycles<br> |
| | + | 72℃ 1min<br> |
| | + | 72℃ 10min<br> |
| | + | 4℃ preservation<br> |
| | + | End<br> |
| | + | DA-PCR splicing reaction product was detected in 2% agarose gel electrophoresis,<br> |
| | + | DA-PCR product 5μl <br> |
| | + | 10×Loading Buffer 0.6μl spotting after mixed<br> |
| | + | 100bp Marker 5μl spotting directly<br> |
| | + | 75V electrophoress for 1h。 </p> |
| | | | |
| - | <p > 94℃ 2min </p> | + | < div style="text-align:center"><p id="coop">< img src="https:// static.igem.org/ mediawiki/ 2014/ 8/ 84/ Simonsong- result-1. png" ></p></ div> |
| - | <p > 94℃ 30S </p>
| + | |
| - | <p > 47℃ 30S 25个循环 </p>
| + | |
| - | <p > 72℃ 1min </p>
| + | |
| - | <p > 72℃ 10min </p>
| + | |
| - | <p > 4℃ 保存 </p>
| + | |
| - | <p > End </p>
| + | |
| - | <p >DA-PCR拼接反应产物检测采用2％的琼脂糖凝胶电泳，其中 </p>
| + | |
| - | <p >DA-PCR产物 5μl </p>
| + | |
| - | <p >10×Loading Buffer 0.6μl 混合均匀后点样 </p>
| + | |
| - | <p >100bp Marker 5μl 直接点样 </p>
| + | |
| - | <p >75V电泳1h。 </p>
| + | |
| - | <p >	 </p>
| + | |
| | | | |
| | + | <p id="coop" align="center">Figure1 DA-PCR splicing results <br></P> |
| | + | <p id="coop"> Illustration of the outcome:<br> |
| | + | 1、A1, A2, A3, A4 could getintermediates (Block1-4) which were made by six single-stranded oligonucleotides splicedtogether, but the A3 and A4 groups could also see by-products which were made by four single-stranded oligonucleotides splicedtogether , and to be made a whole one after recycling of Agarose gel.<br> |
| | + | 2、A5 could obtainintermediates(Block5) which were made by four single-stranded oligonucleotides splicedtogether.</p> |
| | + | <h3 id="coop">4、OE-PCR </h3> |
| | + | <p id="coop">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 id="coop">Block1 1μl<br> |
| | + | Block2 1μl<br> |
| | + | Block3 1μl<br> |
| | + | Block4 1μl<br> |
| | + | Block5 1μl<br> |
| | + | A1 1μl<br> |
| | + | A4 1μl<br> |
| | + | <em>Pfu</em> DNA Polymerase 2.5U 0.5μl <br> |
| | + | dNTP 4μl<br> |
| | + | 10×<em>Pfu</em> buffer（Mg2+） 5μl<br> |
| | | | |
| | | | |
| | | | |
| | | | |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/8/84/Simonsong-result-1.png" ></p> | + | Sterilized ultrapure water to 50ul<br> |
| | + | Procedure of OE-PCR： <br> |
| | + | 94℃ 2min<br> |
| | + | 94℃ 30S<br> |
| | + | 47℃ 30S 20 cycles<br> |
| | + | 72℃ 2min<br> |
| | + | 72℃ 10min<br> |
| | + | 4℃ preservation<br> |
| | + | End<br> |
| | + | The full-length gene spliced by OE-PCR was detected by a 1% agarose gel electrophoresis, specific programs are as follows<br> |
| | + | OE-PCR product 5μl <br> |
| | + | 10×Loading Buffer 0.6μl spotted after mixing well<br> |
| | + | 100bp Marker 5μl spotted directly<br> |
| | + | 80V electrophoresis for 1h.</p> |
| | + | <div style="text-align:center"><p id="coop"><img src="https://static.igem.org/mediawiki/2014/ 2/ 24/Simonsong-result- 2.png" ></p> |
| | | | |
| | + | </div> |
| | | | |
| | | | |
| | | | |
| | | | |
| - | <p >图1 DA-PCR拼接结果 </p> | + | <p id="coop">Illustration of the outcome:</p> |
| - | <p >结果说明： </p> | + | <ol id="coop"> |
| - | <p >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。 </p> | + | <li>L1, L2, L3 could get mlrA genome which were made by five single-stranded oligonucleotides splicedtogether.</li> |
| - | <p >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）。 </p> | + | <li>Wide strip of the tape at 100bp was caused by excessive amount of amplification primers A1 and A4.</li> |
| - | <p > </p>
| + | </ol> |
| - | <h3 >4、OE-PCR </h3> | + | <h3 id="coop">5、Constructing and sequencing of subcoloning vector</h3> |
| - | <p >以DA-PCR拼接出来的的5条双链中间片段（Block）为模版，进一步利用OE-PCR进行连接，OE-PCR的反应体系组成为： </p> | + | <p id="coop">After double enzyme digestion reaction at 37℃ for 3h by<em> Eco</em>RⅠand <em>Pst</em>Ⅰ, MlrA gene and pSB1C3 vector linked together at 16℃ overnight. And then transformed to<em> E.coli</em> JM109 competent cell, and select the desirable colony by using “blue-white selection” method. <br> |
| - | <p > Block1 1μl </p> | + | Constitute of <strong><em>Eco</em>R</strong><strong>Ⅰ</strong><strong>and <em>Pst</em></strong><strong>Ⅰdouble enzyme digestion reaction system</strong><strong>：</strong><strong> </strong><br> |
| - | <p > Block2 1μl </p> | + | Gene or plasmid 10μl<br> |
| - | <p > Block3 1μl </p> | + | <em>Eco</em>RⅠ 0.4μl<br> |
| - | <p > Block4 1μl </p> | + | <em>Pst</em>Ⅰ 0.2μl<br> |
| - | <p > Block5 1μl </p> | + | 10×NEBuffer 3.1 2μl<br> |
| - | <p > A1 1μl </p> | + | sterilizing ulturapure water top up to 20μl<br> |
| - | <p > A4 1μl </p> | + | <strong>Constitute of linking reaction system</strong><strong>：</strong><strong> </strong><br> |
| - | <p > Pfu DNA Polymerase 2.5U 0.5μl </p> | + | mlrA Gene 10μl<br> |
| - | <p > dNTP 4μl </p> | + | pSB1C3 3μl<br> |
| - | <p > 10×Pfu buffer（Mg2+） 5μl </p> | + | T4 ligase 0.5μl<br> |
| - | <p ><img width="349" height="2" src="3_wpsE664.tmp.png" > 灭菌超纯水 补齐至 50μl </p> | + | 10×T4 ligase Buffer 2μl<br> |
| - | <p >OE-PCR反应程序为： </p> | + | sterilizing ulturapure water top up to 20μl<br> |
| - | <p > 94℃ 2min </p> | + | Preparation process of competent cell <strong><em>E.coli</em>BL21</strong><strong>：</strong><strong> </strong><br> |
| - | <p ><img width="13" height="64" src="3_wpsE675.tmp.png" > 94℃ 30S </p> | + | (1) Culturing strain <em>E.coli</em>BL21(37℃)overnight to recovery culture, Adding overnight bacteria culture fluid 600uLto 30 mL LB culture medium, and 37℃，200 r/min shake culturing 1-2 h, until OD600 is around 0.3-0.4.<br> |
| - | <p > 47℃ 30S 20个循环 </p> | + | (2) In the aseptic condition, transfer 30mLbacteria fluid to the centrifugal tube in the ice 10 min to cooling the culture to 0℃.<br> |
| - | <p > 72℃ 2min </p> | + | (3) Centrifuging 10 min in precooling centrifugal machine over 4000r/min, throw supernate, and then add30mLprecooling 0.1mol/L CaCl2 solution to resuspension precipitation in the ice 10 min.<br> |
| - | <p > 72℃ 10min </p> | + | (4) Centrifuging 4000r/min in 4℃10min, throw supernate, each 30mL initial culture resuspension cell precipitation with 1.0mL precooling 0.1mol/LCaCl2 solution, and then 200μL each one.<br> |
| - | <p ><img width="349" height="2" src="3_wpsE686.tmp.png" > 4℃ 保存 </p> | + | <strong>Heal shock transformation process</strong><strong>：</strong><strong> </strong><br> |
| - | <p > End </p> | + | (1) Thawing freshly prepared or -80℃ competentcellBL21suspension.<br> |
| - | <p >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 </p> | + | (2) Add constructed recombinant cloning vector plasmidspSB1C3-A(no more than 50ng, 10μl), shake gently, and then in the ice 30min.<br> |
| - | <p ><img width="13" height="34" src="3_wpsE687.tmp.png" >OE-PCR产物 5μl </p> | + | (3) Put EP tube in 42℃ water bath90s，and then transfer it to ice water bath to cool cells1-2 min.<br> |
| - | <p >10×Loading Buffer 0.6μl 混合均匀后点样 </p> | + | (4) add 800μL LB medium to EP tube，transfer it to 37℃ table，150r/min，to recovery strain 45 min.<br> |
| - | <p ><img width="349" height="2" src="3_wpsE688.tmp.png" >100bp Marker 5μl 直接点样 </p> | + | Spread plate and Culture<br> |
| - | <p >80V电泳1h。 </p> | + | (1)After culture in 37℃, centrifuge the cell solution(5000rpm,5min) and concentrate it to 200μl. Then coat it to LB culture with chloramphenicol and set a positive control.<br> |
| - | <p > </p> | + | (2) Wait until the solution in culture is full absorbed by cell, then invert the plate and culture it in 37℃ overnight.<br> |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/2/24/Simonsong-result-2.png" ></p> | + | Sequence test<br> |
| | + | Replace the fragment(45bp) between EcoR I site and Pst I site with mlrA(1024bp) on pSB1C3 vector and then we get the reconstructed plasmid, pSB1C3-A.</p> |
| | | | |
| | | | |
| | | | |
| | | | |
| | + | <div style="text-align:center"><p id="coop"><img src="https://static.igem.org/mediawiki/2014/0/0d/Simonsong-result-3.png" > </p></div> |
| | | | |
| | | | |
| - | <p >图2 OE-PCR拼接结果 </p>
| |
| | | | |
| | | | |
| | + | <p id="coop" align="center">Fig3.Reconstructed vector pSB1C3-A </p> |
| | + | <p id="coop">Select the white colony on culture and culture it. Then we extract the plasmid and use it as template in PCR to identify the colony. In theory, the reconstructed vector with mlrA fragment is 1293bp and the empty vector without mlrA fragment is 314bp. So, the result shows that the white colony we select has reconstructed vector with mlrA fragment in it.</p> |
| | | | |
| | + | <div style="text-align:center"><p id="coop"><img src="https://static.igem.org/mediawiki/2014/6/63/Simonsong-result-4.png" ></p></div> |
| | | | |
| | | | |
| | | | |
| | + | <p id="coop" align="center"> Fig 4. The identification of reconstructed vector pSB1C3-A </p> |
| | + | <p id="coop">Results: </p> |
| | + | <p id="coop">1.L1 and L2 is 1300bp and it confirms that the reconstructed vector has mlrA on it. </p> |
| | + | <p id="coop">2.M is the 100bp Ladde Marker. </p> |
| | + | <p id="coop">Sequencing result shows that the sequence of synthetic mlrA gene is exactly same as sequence of designed sequence. </p> |
| | | | |
| - | <p >结果说明： </p>
| |
| - | <p >1、L1、L2、L3都能得到5条中间产物拼接到一起的mlrA全基因。 </p>
| |
| - | <p >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。 </p>
| |
| | | | |
| | | | |
| | | | |
| | + | <div style="text-align:center"><p id="coop"><img src="https://static.igem.org/mediawiki/2014/5/5a/Simonsong-result-5.png" ><img src="https://static.igem.org/mediawiki/2014/0/0b/Simonsong-result-6.png" > </p></div> |
| | + | <p id="coop"align="center" > Fig5.Comparison of result of sequencing and designed sequence </p> |
| | + | <p id="coop"> </p> |
| | | | |
| - | <h3 >5、亚克隆载体构建与测序 </h3>
| |
| - | <p >合成的mlrA基因与pSB1C3载体经过EcoRⅠ和PstⅠ于37℃双酶切3h后，16℃连接过夜，转化到E.coli JM109感受态细胞中，经蓝白筛选挑取白色菌落。 </p>
| |
| - | <p >EcoRⅠ和PstⅠ双酶切反应体系组成为： </p>
| |
| - | <p > Gene or plasmid 10μl </p>
| |
| - | <p > EcoRⅠ 0.4μl </p>
| |
| - | <p > PstⅠ 0.2μl </p>
| |
| - | <p > 10×NEBuffer 3.1 2μl </p>
| |
| - | <p > 灭菌超纯水 补齐至 20μl </p>
| |
| - | <p >连接反应体系组成为： </p>
| |
| - | <p > mlrA Gene 10μl </p>
| |
| - | <p > pSB1C3 3μl </p>
| |
| - | <p > T4 ligase 0.5μl </p>
| |
| - | <p > 10×T4 ligase Buffer 2μl </p>
| |
| - | <p > 灭菌超纯水 补齐至 20μl </p>
| |
| - | <p >E.coli JM109感受态细胞制备流程： </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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℃。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >热激转化过程： </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >平板涂布及培养过程： </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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； </p>
| |
| - | <p >实验连接组：连接产物，氯霉素板 </p>
| |
| - | <p >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 </p>
| |
| - | <p >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。 </p>
| |
| - | <p >空菌组：感受态细胞，无氯霉素板 </p>
| |
| - | <p >序列测定 </p>
| |
| - | <p >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）。 </p>
| |
| | | | |
| | | | |
| - | | + | <div style="background:#FEE5AD;"> |
| - | | + | <a height="30px" width="5%" align="center" onMouseOver="this.bgColor='#FFFFFF'" onMouseOut="this.bgColor='#A1DBB2'" href="#menu" style="text-decoration:none;color:#1C140D;float:right;background:#A1DBB2;"> Top </a> |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/0/0d/Simonsong-result-3.png" > </p> | + | </div> |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | <p >图3 重组载体pSB1C3-A </p>
| + | |
| - | <p >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。 </p>
| + | |
| - | | + | |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/6/63/Simonsong-result-4.png" ></p>
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | <p > 图4 重组载体pSB1C3-A的PCR鉴定 </p> | + | |
| - | <p >结果说明： </p>
| + | |
| - | <p >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 </p>
| + | |
| - | <p >2、M是100bp Ladder Marker </p>
| + | |
| - | <p >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。 </p>
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/5/5a/Simonsong-result-5.png" ><img src="https://static.igem.org/mediawiki/2014/0/0b/Simonsong-result-6.png" > </p>
| + | |
| - | <p >图5 测序结果与设计序列比对 </p>
| + | |
| - | <p > </p>
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | <h3 ><img width="155" height="58" src="6_wps9D1F.tmp.png" >6、引物合成 </h3>
| + | |
| - | <table >
| + | |
| - | <tr >
| + | |
| - | <td width="1038" valign="center" rowspan="3" ><p > </p>
| + | |
| - | <table >
| + | |
| - | <tr >
| + | |
| - | <td width="1023" valign="center" rowspan="2" ><p >吉林省库美生物科技有限公司引物合成订购单 </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr ></tr>
| + | |
| - | </table>
| + | |
| - | </table>
| + | |
| - | <p > </p>
| + | |
| - | <table >
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >引物编号 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >序列，(5' to 3') </p></td>
| + | |
| - | <td width="57" valign="center" ><p >碱基数（bp） </p></td>
| + | |
| - | <td width="57" valign="center" ><p >合成总量(OD) </p></td>
| + | |
| - | <td width="47" valign="center" ><p >分装管数 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >纯化方式 </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A101 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A102 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CTCGTAAAGCATGAGCAGCTAAAGCAATTAAAATAGCTAAAACATAAAAACATAATA </p></td>
| + | |
| - | <td width="57" valign="center" ><p >57 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A103 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >AGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A104 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >AGCAGTAATAATATTTAAATGAGCATGAGTTTCTTGTAACATTTTAAACATTGGATCTAA </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A105 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A106 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CAGCAAACATTGGAGCAGCTGGAAATAATAATAAAGTATAAGCTCCTGGATATTCA </p></td>
| + | |
| - | <td width="57" valign="center" ><p >56 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A107 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >GCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A108 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >TCGCCATGGAGCACATCGTGATAATAATTCTCGAAATCCAGCTTGTCCATATCCAA </p></td>
| + | |
| - | <td width="57" valign="center" ><p >56 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A109 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >ACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A110 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CATAATTCCAGTTAAAGCAAAAAAAGCTAAAAAACAAACAGCAATAACAGTAACTCCTTG </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A111 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >TTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A112 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CAACTGGATCTGATCCATATCGTAAAAAAGTTCGATCTAAAGTTCCTGGTGGAGCATATA </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A113 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCA </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A114 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAGCAGCT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >44 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A201 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCC </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A202 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CACCACATAATTCCTAAAATAACAGCAGCAGTTAATGGATCAAATTTTTTTAATAATTGT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A203 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >TGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A204 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >AACTGACCAAGCAGCTCCTGGAGCTCCTGAAAATAAAGTTGGTAAATCTCGTGGT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >55 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A205 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >AGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTC </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A206 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CCTAATTTATTACAAACAAAAACAGCAATAATAGTTGAAGCAATAAATCCTGGAGTAATA </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A207 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >TGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGC </p></td>
| + | |
| - | <td width="57" valign="center" ><p >57 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A208 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CATTCAGCAGTAACATTAACTCCTAATTCATTATGAATAGCATGAGTTAAAACTCCTCCC </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A209 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >AGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGA </p></td>
| + | |
| - | <td width="57" valign="center" ><p >55 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A210 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >ACTAATCCAATAGCAACAGCAAATTCAATTAAATCCCATGGTCGCCATCCTAATCCAGCA </p></td>
| + | |
| - | <td width="57" valign="center" ><p >60 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A211 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >ATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTC </p></td>
| + | |
| - | <td width="57" valign="center" ><p >57 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A212 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >GTGGAACATTTCCCCAAGCTAATCGAGCATTATCTGGTGAAGCAGCTCCTAATGATCGT </p></td>
| + | |
| - | <td width="57" valign="center" ><p >59 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A213 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >TTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGC </p></td>
| + | |
| - | <td width="57" valign="center" ><p >59 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A214 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >AAGACGTCCTATTAAGCATTAGCTCCTGATTTATCTCCAACTC </p></td>
| + | |
| - | <td width="57" valign="center" ><p >43 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A1 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >CCGGAATTCATGCGAGAATTTGTTCG </p></td>
| + | |
| - | <td width="57" valign="center" ><p >26 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A2 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAG </p></td>
| + | |
| - | <td width="57" valign="center" ><p >39 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A3 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >ATTAGAAGAATTAGGATGGCGAGGATTTGC </p></td>
| + | |
| - | <td width="57" valign="center" ><p >30 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A4 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >AAGACGTCCTATTAAGCATTAGCTCCTG </p></td>
| + | |
| - | <td width="57" valign="center" ><p >28 </p></td>
| + | |
| - | <td width="57" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="47" valign="center" ><p >1 </p></td>
| + | |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| + | |
| - | </tr>
| + | |
| - | <tr >
| + | |
| - | <td width="50" valign="center" ><p >A5 </p></td>
| + | |
| - | <td width="456" valign="center" ><p >AACTGCAGCTATTAAGCATTAGCTCCTG </p></td>
| + | |
| - | <td width="57" valign="center" ><p >28 </p></td>
| + | |
| - | <td width="57" valign="center" ><p > </p></td>
| + | |
| - | <td width="47" valign="center" ><p > </p></td>
| + | |
| - | <td width="56" valign="center" ><p > </p></td>
| + | |
| - | </tr>
| + | |
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| + | |
| - | </table> | + | |
| | </div> | | </div> |
| | <td width="100%" rowspan="3" align="left"> | | <td width="100%" rowspan="3" align="left"> |
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| - | .observation{}
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| - | #coop{font-size:18px;padding:0px 20px;}
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| - | .home_second_block_div{width:265px;
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| - | </style>
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| - | </head>
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| - | <style>
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| - | #contentSub, #footer-box, #catlinks, #search-controls, #p-logo, .printfooter, .firstHeading,.visualClear {display: none;} /*-- hides default wiki settings --*/
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| - | </style>
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| - | <body style="width:965px;display:block;word-break: break-all;word-wrap: break-word;margin:0 auto;">
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| - |
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| - | <!-- menu -->
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| - | <div class="menu">
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| - | <!---吉大logo---->
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| - | <img style="height:80px;"src="https://static.igem.org/mediawiki/2014/a/a3/Uea_igem-logo.png">
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| - | <!---比赛logo---->
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| - | <img style="height:80px;"src="https://static.igem.org/mediawiki/2014/6/60/Jilin_China_igem_logo.gif">
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| - | <table id="menu" cellspacing="0" height="135px">
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| - | <ul>
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| - | <li><a style="margin-top:20px;" class="hide" href="https://2014.igem.org/Team:Jilin_China">HOME</a></li>
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| - | <li><a style="margin-top:20px;" class="hide">TEAM<span class="caret"></span></a>
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| - | <ul>
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| - | <li><a style="margin-top:20px;" href="https://2014.igem.org/Team:Jilin_China/Team Members" title="Cross browser fixed layout">OUR TEAM</a></li>
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| - | <li><a href="https://2014.igem.org/Team:Jilin_China/ATTRIBUTION" title="Cross browser fixed layout">ATTRIBUTION</a></li>
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| - | </ul>
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| - | </li>
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| - |
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| - | <li><a style="margin-top:20px;" class="hide" >PROJECT<span class="caret"></span></a>
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| - | <ul>
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| - | <li><a style="margin-top:20px;" href="https://2014.igem.org/Team:Jilin_China/NOTEBOOK" title="a coded list of spies">NOTEBOOK</a></li>
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| - | <li><a href="https://2014.igem.org/Team:Jilin_China/RESULT" title="a horizontal vertical menu">RESULT</a></li>
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| - | </ul>
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| - |
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| - | <li><a style="margin-top:20px;" class="hide">IDEA<span class="caret"></span></a>
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| - | <li><a style="margin-top:20px;" href="https://2014.igem.org/Team:Jilin_China/BACKGROUND" title="A drop down menu">BACKGROUND</a></li>
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| - | <li><a href="https://2014.igem.org/Team:Jilin_China/DESIGN" title="A cascading menu">DESIGN</a></li>
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| - | </ul>
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| - | <li><a style="margin-top:20px;" class="hide">PRACTICE<span class="caret"></span></a>
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| - | <li><a style="margin-top:20px;" href="https://2014.igem.org/Team:Jilin_China/Coorperation" title="colour wheel">COORPERATION</a></li>
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| - | <li><a href="https://2014.igem.org/Team:Jilin_China/observation" title="a menu using opacity">OBSERVATION</a></li>
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| - | <li><a href="https://2014.igem.org/Team:Jilin_China/Propagate" title="partial opacity">PROPAGATE</a></li>
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| - | <li><a href="https://2014.igem.org/Team:Jilin_China/Survey" title="partial opacity II">SURVEY</a></li>
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| - | </ul>
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| - | </li>
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| - |
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| - | <li><a style="margin-top:20px;" class="hide" href="https://2014.igem.org/Team:Jilin_China/Acknowledgement">ACKNOWLEDGEMENT</a></li>
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| - | <!-- clear the floats if required -->
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| - | <td background="https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png"></td>
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| - |
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| - | <!-- You can replace this image with your team's logo! -->
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| - |
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| - | <h3>Welcome! <br>Team Jilin China</h3>
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| - | </td>
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| - | <td background="https://static.igem.org/mediawiki/2014/d/d7/Uea_body-bg.png"></td>
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| - | </tr>
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| - | </table>
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| - | <table width="100%" cellspacing="0" height="0px">
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| - | <tr><td bgColor="#FEE5AD"></td>
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| - | <td width="100%" align="left" bgColor="#FEE5AD" >
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| - | <table width="975px" cellspacing="0" height="0px">
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| - | <tr>
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| - | <div class="observation"><center><h3>mlrA基因合成</h3></center> </div>
| |
| - | <p >一、实验方案 </p>
| |
| - | <p >1、目的蛋白氨基酸序列 </p>
| |
| - | <p >MlrA GenBank: AF411068	SOURCE Sphingomonas sp. ACM-3962 </p>
| |
| - | <p >MREFVRQRPLLCFYVLAILIALAAHALRAMSPTPLDPMFKMLQETHAHLNIITAVRSTFEYPGAYTLLLFPAAPMFAALIATGIGYGQAGFRELLSRCAPWRSPVSWRQGVTVIAVCFLAFFALTGIMWVQTYLYAPPGTLDRTFLRYGSDPVAIYVMLAASLLLSPGPLLEELGWRGFALPQLLKKFDPLTAAVILGIMWWAWHLPRDLPTLFSGAPGAAWSVIVKQLVITPGFIASTIIAVFVCNKLGGSMWGGVLTHAIHNELGVNVTAEWAPTVAGLGWRPWDLIEFAVAIGLVLICGRSLGAASPDNARLAWGNVPPKLPGGVGDKSGANA </p>
| |
| - | <h3 >2、密码子选择 </h3>
| |
| - | <p >Lactococcus lactis subsp. cremoris SK11 [gbbct]: 2504 CDS's (696252 codons) </p>
| |
| - | <p >http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272622 </p>
| |
| - |
| |
| - | <p >fields: [triplet] [frequency: per thousand] ([number]) </p>
| |
| - | <p >UUU 36.1( 25117) UCU 16.5( 11473) UAU 28.4( 19782) UGU 3.9( 2729) </p>
| |
| - | <p >UUC 12.0( 8326) UCC 3.3( 2266) UAC 8.1( 5665) UGC 1.1( 771) </p>
| |
| - | <p >UUA 31.0( 21599) UCA 21.1( 14698) UAA 2.5( 1708) UGA 0.7( 498) </p>
| |
| - | <p >UUG 21.2( 14782) UCG 3.9( 2685) UAG 0.4( 298) UGG 10.1( 7000) </p>
| |
| - | <p > </p>
| |
| - | <p >CUU 25.2( 17574) CCU 11.9( 8253) CAU 13.3( 9269) CGU 14.6( 10159) </p>
| |
| - | <p >CUC 8.1( 5614) CCC 2.9( 1990) CAC 4.5( 3121) CGC 4.5( 3122) </p>
| |
| - | <p >CUA 8.0( 5591) CCA 14.5( 10061) CAA 31.1( 21678) CGA 5.9( 4140) </p>
| |
| - | <p >CUG 6.4( 4476) CCG 2.9( 2010) CAG 6.6( 4596) CGG 2.3( 1570) </p>
| |
| - | <p > </p>
| |
| - | <p >AUU 51.0( 35505) ACU 20.4( 14218) AAU 40.1( 27903) AGU 14.7( 10220) </p>
| |
| - | <p >AUC 16.1( 11197) ACC 7.4( 5135) AAC 11.1( 7709) AGC 6.2( 4328) </p>
| |
| - | <p >AUA 8.7( 6023) ACA 22.1( 15386) AAA 61.2( 42611) AGA 8.0( 5557) </p>
| |
| - | <p >AUG 24.7( 17203) ACG 7.1( 4919) AAG 13.5( 9375) AGG 1.7( 1152) </p>
| |
| - | <p > </p>
| |
| - | <p >GUU 30.8( 21415) GCU 30.1( 20949) GAU 37.3( 25965) GGU 23.4( 16287) </p>
| |
| - | <p >GUC 12.7( 8841) GCC 12.1( 8400) GAC 14.4( 10018) GGC 8.5( 5906) </p>
| |
| - | <p >GUA 12.8( 8921) GCA 22.3( 15521) GAA 56.1( 39035) GGA 24.5( 17046) </p>
| |
| - | <p >GUG 9.2( 6376) GCG 8.2( 5708) GAG 13.0( 9070) GGG 8.2( 5732) </p>
| |
| - | <p >Coding GC 36.75% 1st letter GC 48.61% 2nd letter GC 34.45% 3rd letter GC 27.20% </p>
| |
| - | <p >密码子密码子密码子 </p>
| |
| - | <p > </p>
| |
| - | <p >使用密码子表 </p>
| |
| - | <p >* TAA </p>
| |
| - | <p >A GCT </p>
| |
| - | <p >C TGT </p>
| |
| - | <p >D GAT </p>
| |
| - | <p >E GAA </p>
| |
| - | <p >F TTT </p>
| |
| - | <p >G GGA </p>
| |
| - | <p >H CAT </p>
| |
| - | <p >I ATT </p>
| |
| - | <p >K AAA </p>
| |
| - | <p >L TTA </p>
| |
| - | <p >M ATG </p>
| |
| - | <p >N AAT </p>
| |
| - | <p >P CCA </p>
| |
| - | <p >Q CAA </p>
| |
| - | <p >R CGA </p>
| |
| - | <p >S TCA </p>
| |
| - | <p >T ACT </p>
| |
| - | <p >V GTT </p>
| |
| - | <p >W TGG </p>
| |
| - | <p >Y TAT </p>
| |
| - | <p > </p>
| |
| - | <h3 >3、逆翻译序列及限制性内切酶选择 </h3>
| |
| - | <p >EcoR I </p>
| |
| - | <p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGCTGCAGTT </p>
| |
| - |
| |
| - |
| |
| - | <h3 >4、序列分析 </h3>
| |
| - | <p >_Base Count : 1008 bp (270 A, 377 T, 158 C, 203 G)<br>
| |
| - |  _Composition : 36% GC, 64% AT<br>
| |
| - | <br>
| |
| - | <img width="101" height="27" src="file:///C:\Users\ADMINI~1\AppData\Local\Temp\ksohtml\wps7345.tmp.wmf" ></p>
| |
| - | <p >Absent Sites<br>
| |
| - | AarI AatII Acc65I AccI AciI AclI AcuI AfeI AflII AflIII AgeI AhdI AleI ApaI ApaLI AscI AseI AsiSI AsuII AvaI AvrII BaeGI BamHI BanI BbsI BbvCI BceAI BciVI BclI BfaI BfuAI BglI BglII BlpI BmgBI BmrI BmtI Bpu10I BpuEI BsaAI BsaHI BsaI BsaWI BseMII BseYI BsgI BsiEI BsiWI BslI BsmAI BsmBI BsmFI BsmI BsoBI BspCNI BspEI BspHI BspMI BspQI BsrBI BsrDI BsrFI BsrGI BssHII BstAPI BstBI BstEII BstUI BstZ17I Bsu36I BtgZI BtrI BtsI Cac8I ClaI CviQI DdeI DrdI EaeI EagI EarI EciI Eco31I EcoNI EcoO109I EcoRI EcoRV Esp3I FauI FseI FspAI FspI HaeII HaeIII HgaI HhaI HinP1I HincII HindIII HinfI HpaI HpaII Hpy99I HpyAV HpyCH4IV HpyCH4V KasI KpnI MaeI MaeII MfeI MluI MlyI MscI NaeI NarI NciI NdeI NgoMIV NheI NmeAIII NotI NruI NsiI NspI PacI PasI PciI PflFI PflMI PleI PmeI PpuMI PshAI PsiI PspOMI PspXI PstI PvuI RsaI RsrII SacII SalI SanDI SbfI ScaI SexAI SfaNI SfcI SfiI SfoI SgrAI SmaI SmlI SnaBI SpeI SphI SrfI StuI TaiI TatI TauI TfiI TspGWI TspMI TspRI Tth111I XbaI XhoI XmaI ZraI </p>
| |
| - | <p >Unique Sites </p>
| |
| - | <p >XmnI (6) MslI (25) </p>
| |
| - | <p >Tsp45I (89) </p>
| |
| - | <p >SwaI (142) SspI (147) </p>
| |
| - | <p >MspA1I (211) </p>
| |
| - | <p >DraIII (286) </p>
| |
| - | <p >BstXI (309) </p>
| |
| - | <p >BsaBI (439) Hpy188I (447) </p>
| |
| - | <p >Sau96I (501) </p>
| |
| - | <p >MboII (513) </p>
| |
| - | <p >BssSI (619) </p>
| |
| - | <p >SacI (646) XcmI (651) AlwNI (652) </p>
| |
| - | <p >NlaIV (821) </p>
| |
| - | <p >Hpy166II (901) </p>
| |
| - | <h3 >5、序列拆分 </h3>
| |
| - | <p >The maximum allowable assembly oligo length is equal to the target assembly oligo length (60). This may cause some weird behavior, especially in terms of overlap melting temperature. </p>
| |
| - | <p > </p>
| |
| - | <p >2 building blocks were generated. </p>
| |
| - | <p >Building Block .1   529bp   1..529 <br>
| |
| - | Left  - 5' CCGGAATTCATGCGAGAATTTGTTCG 3'<br>
| |
| - | Rght  - 5' ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAG 3'<br>
| |
| - | RghtU - 5' ATTCTTCTAAUAATGGTCCTGGTGATAATAATAATGAAG 3'<br>
| |
| - | Sequence: </p>
| |
| - | <p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAAT </p>
| |
| - | <p > </p>
| |
| - | <p >Assembly Oligos: average overlap Tm is 47°;average oligo length is 58bp. </p>
| |
| - | <p > </p>
| |
| - | <p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAAT<br>
| |
| - | CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCT              AGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTT              CTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTT              GCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTT              ACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGT               TTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTT                CGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCA                      <br>
| |
| - |                                      ATAATACAAAAATACAAAATCGATAAAATTAACGAAATCGACGAGTACGAAATGCTC                 AATCTAGGTTACAAATTTTACAATGTTCTTTGAGTACGAGTAAATTTATAATAATGACGA              ACTTATAGGTCCTCGAATATGAAATAATAATAAAGGTCGACGAGGTTACAAACGAC                  AACCTATACCTGTTCGACCTAAAGCTCTTAATAATAGTGCTACACGAGGTACCGCT                  GTTCCTCAATGACAATAACGACAAACAAAAAATCGAAAAAAACGAAATTGACCTTAATAC                ATATACGAGGTGGTCCTTGAAATCTAGCTTGAAAAAATGCTATACCTAGTCTAGGTCAAC                TCGACGAAGTAATAATAATAGTGGTCCTGGTAATAATCTTCTTA<br>
| |
| - | GGCCTTAAGTACGCTCTTAAACAAGCTGTTGCTGGTAATAATACAAAAATACAAAATCGATAAAATTAACGAAATCGACGAGTACGAAATGCTCGATACAGTGGTTGAGGTAATCTAGGTTACAAATTTTACAATGTTCTTTGAGTACGAGTAAATTTATAATAATGACGACAAGCTAGTTGAAAACTTATAGGTCCTCGAATATGAAATAATAATAAAGGTCGACGAGGTTACAAACGACGAAATTAACGATGACCTTAACCTATACCTGTTCGACCTAAAGCTCTTAATAATAGTGCTACACGAGGTACCGCTAGTGGTCAAAGTACCGCTGTTCCTCAATGACAATAACGACAAACAAAAAATCGAAAAAAACGAAATTGACCTTAATACACCCAAGTTTGAATAAATATACGAGGTGGTCCTTGAAATCTAGCTTGAAAAAATGCTATACCTAGTCTAGGTCAACGATAAATACAATACAATCGACGAAGTAATAATAATAGTGGTCCTGGTAATAATCTTCTTA  </p>
| |
| - | <p > </p>
| |
| - | <p >Building Block .2   513bp   519..1031 <br>
| |
| - | Left  - 5' ATTAGAAGAATTAGGATGGCGAGGATTTGC 3'<br>
| |
| - | LeftU - 5' ATTAGAAGAAUTAGGATGGCGAGGATTTGC 3'<br>
| |
| - | Rght  - 5' AAGACGTCCTATTAAGCATTAGCTCCTG 3'<br>
| |
| - | Sequence: </p>
| |
| - | <p >ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGGACGTCTT </p>
| |
| - | <p >Assembly Oligos: average overlap Tm is 49°;average oligo length is 58bp. </p>
| |
| - | <p >ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGGACGTCTT<br>
| |
| - | ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCC            TGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATT            AGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTC            TGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGC               AGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGA                 ATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTC                TTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGC                     <br>
| |
| - |                                     TGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCAC            TGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAA                 ATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCC            CCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTAC            ACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCA            TGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTG               CTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA<br>
| |
| - | TAATCTTCTTAATCCTACCGCTCCTAAACGAAATGGTGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCACCCGAACCGTAAATGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAATAACAATTTGTTAATCAATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCCTCCTAGTTACACCCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTACCCGAGGTTGACAACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCAAAATTAAACACCTGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTGGTTTTAATGGTCCTCCTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA </p>
| |
| - |
| |
| - |
| |
| - | <h2 >二、实验操作及结果分析 </h2>
| |
| - | <h3 >1、引物溶解 </h3>
| |
| - | <p >根据合成引物的分子量，计算需要添加的灭菌去离子水的量，将合成的引物稀释成10um/L备用。 </p>
| |
| - | <table align="center" >
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >引物编号 </p></td>
| |
| - | <td width="48" valign="center" ><p >Tm </p></td>
| |
| - | <td width="85" valign="center" ><p >MW(g/mole) </p></td>
| |
| - | <td width="74" valign="center" ><p >nmol/Tube </p></td>
| |
| - | <td width="177" valign="center" ><p >Add buffer for 10μM/uL </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A101 </p></td>
| |
| - | <td width="48" valign="center" ><p >68.3 </p></td>
| |
| - | <td width="85" valign="center" ><p >18451 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >155 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A102 </p></td>
| |
| - | <td width="48" valign="center" ><p >65 </p></td>
| |
| - | <td width="85" valign="center" ><p >17588.4 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.4 </p></td>
| |
| - | <td width="177" valign="center" ><p >142 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A103 </p></td>
| |
| - | <td width="48" valign="center" ><p >69.7 </p></td>
| |
| - | <td width="85" valign="center" ><p >18359 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.5 </p></td>
| |
| - | <td width="177" valign="center" ><p >155 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A104 </p></td>
| |
| - | <td width="48" valign="center" ><p >64.2 </p></td>
| |
| - | <td width="85" valign="center" ><p >18533 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.4 </p></td>
| |
| - | <td width="177" valign="center" ><p >143 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A105 </p></td>
| |
| - | <td width="48" valign="center" ><p >66.3 </p></td>
| |
| - | <td width="85" valign="center" ><p >18354 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >155 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A106 </p></td>
| |
| - | <td width="48" valign="center" ><p >68 </p></td>
| |
| - | <td width="85" valign="center" ><p >17321.2 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.5 </p></td>
| |
| - | <td width="177" valign="center" ><p >151 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A107 </p></td>
| |
| - | <td width="48" valign="center" ><p >71.1 </p></td>
| |
| - | <td width="85" valign="center" ><p >18533 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >156 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A108 </p></td>
| |
| - | <td width="48" valign="center" ><p >70.9 </p></td>
| |
| - | <td width="85" valign="center" ><p >17127.2 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >164 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A109 </p></td>
| |
| - | <td width="48" valign="center" ><p >73.8 </p></td>
| |
| - | <td width="85" valign="center" ><p >18513 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >156 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A110 </p></td>
| |
| - | <td width="48" valign="center" ><p >66.3 </p></td>
| |
| - | <td width="85" valign="center" ><p >18440 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.4 </p></td>
| |
| - | <td width="177" valign="center" ><p >137 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A111 </p></td>
| |
| - | <td width="48" valign="center" ><p >67.6 </p></td>
| |
| - | <td width="85" valign="center" ><p >18426 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >156 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A112 </p></td>
| |
| - | <td width="48" valign="center" ><p >69.7 </p></td>
| |
| - | <td width="85" valign="center" ><p >18506 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.5 </p></td>
| |
| - | <td width="177" valign="center" ><p >147 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A113 </p></td>
| |
| - | <td width="48" valign="center" ><p >67 </p></td>
| |
| - | <td width="85" valign="center" ><p >18441 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >156 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A114 </p></td>
| |
| - | <td width="48" valign="center" ><p >64 </p></td>
| |
| - | <td width="85" valign="center" ><p >13568.8 </p></td>
| |
| - | <td width="74" valign="center" ><p >2.0 </p></td>
| |
| - | <td width="177" valign="center" ><p >200 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A201 </p></td>
| |
| - | <td width="48" valign="center" ><p >66.3 </p></td>
| |
| - | <td width="85" valign="center" ><p >18568 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.4 </p></td>
| |
| - | <td width="177" valign="center" ><p >143 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A202 </p></td>
| |
| - | <td width="48" valign="center" ><p >64.9 </p></td>
| |
| - | <td width="85" valign="center" ><p >18407 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.5 </p></td>
| |
| - | <td width="177" valign="center" ><p >145 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A203 </p></td>
| |
| - | <td width="48" valign="center" ><p >68.3 </p></td>
| |
| - | <td width="85" valign="center" ><p >18522 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.5 </p></td>
| |
| - | <td width="177" valign="center" ><p >155 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A204 </p></td>
| |
| - | <td width="48" valign="center" ><p >71.8 </p></td>
| |
| - | <td width="85" valign="center" ><p >16968 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >162 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A205 </p></td>
| |
| - | <td width="48" valign="center" ><p >68.3 </p></td>
| |
| - | <td width="85" valign="center" ><p >18491 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.5 </p></td>
| |
| - | <td width="177" valign="center" ><p >154 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A206 </p></td>
| |
| - | <td width="48" valign="center" ><p >64.9 </p></td>
| |
| - | <td width="85" valign="center" ><p >18492 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.4 </p></td>
| |
| - | <td width="177" valign="center" ><p >138 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A207 </p></td>
| |
| - | <td width="48" valign="center" ><p >67.9 </p></td>
| |
| - | <td width="85" valign="center" ><p >17753.4 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >156 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A208 </p></td>
| |
| - | <td width="48" valign="center" ><p >68.3 </p></td>
| |
| - | <td width="85" valign="center" ><p >18305 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.5 </p></td>
| |
| - | <td width="177" valign="center" ><p >151 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A209 </p></td>
| |
| - | <td width="48" valign="center" ><p >72.6 </p></td>
| |
| - | <td width="85" valign="center" ><p >17117 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >164 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A210 </p></td>
| |
| - | <td width="48" valign="center" ><p >70.4 </p></td>
| |
| - | <td width="85" valign="center" ><p >18278 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.5 </p></td>
| |
| - | <td width="177" valign="center" ><p >149 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A211 </p></td>
| |
| - | <td width="48" valign="center" ><p >68.6 </p></td>
| |
| - | <td width="85" valign="center" ><p >17622.4 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.7 </p></td>
| |
| - | <td width="177" valign="center" ><p >165 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A212 </p></td>
| |
| - | <td width="48" valign="center" ><p >72.6 </p></td>
| |
| - | <td width="85" valign="center" ><p >18145.8 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >156 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A213 </p></td>
| |
| - | <td width="48" valign="center" ><p >71.9 </p></td>
| |
| - | <td width="85" valign="center" ><p >18356.8 </p></td>
| |
| - | <td width="74" valign="center" ><p >1.5 </p></td>
| |
| - | <td width="177" valign="center" ><p >146 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A214 </p></td>
| |
| - | <td width="48" valign="center" ><p >66.9 </p></td>
| |
| - | <td width="85" valign="center" ><p >13072.6 </p></td>
| |
| - | <td width="74" valign="center" ><p >2.2 </p></td>
| |
| - | <td width="177" valign="center" ><p >220 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A1 </p></td>
| |
| - | <td width="48" valign="center" ><p >62 </p></td>
| |
| - | <td width="85" valign="center" ><p >8002.2 </p></td>
| |
| - | <td width="74" valign="center" ><p >3.6 </p></td>
| |
| - | <td width="177" valign="center" ><p >356 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A2 </p></td>
| |
| - | <td width="48" valign="center" ><p >61 </p></td>
| |
| - | <td width="85" valign="center" ><p >12043.8 </p></td>
| |
| - | <td width="74" valign="center" ><p >2.2 </p></td>
| |
| - | <td width="177" valign="center" ><p >222 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A3 </p></td>
| |
| - | <td width="48" valign="center" ><p >62 </p></td>
| |
| - | <td width="85" valign="center" ><p >9375 </p></td>
| |
| - | <td width="74" valign="center" ><p >2.8 </p></td>
| |
| - | <td width="177" valign="center" ><p >281 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A4 </p></td>
| |
| - | <td width="48" valign="center" ><p >62 </p></td>
| |
| - | <td width="85" valign="center" ><p >8548.6 </p></td>
| |
| - | <td width="74" valign="center" ><p >3.3 </p></td>
| |
| - | <td width="177" valign="center" ><p >330 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="47" valign="center" ><p >A5 </p></td>
| |
| - | <td width="48" valign="center" ><p >62 </p></td>
| |
| - | <td width="85" valign="center" ><p >8548.6 </p></td>
| |
| - | <td width="74" valign="center" ><p >3.3 </p></td>
| |
| - | <td width="177" valign="center" ><p >330 </p></td>
| |
| - | </tr>
| |
| - | </table>
| |
| - | <p > </p>
| |
| - | <h3 >2、引物混合 </h3>
| |
| - | <p >合成的寡核苷酸呈粉末状，使用前需要10000rpm离心1min，使寡核苷酸干粉集中在管底，使用时小心开启瓶盖。 </p>
| |
| - | <p >溶解寡核苷酸前核对合成报告单与引物标签上的OD数是否一致，并核对分装管数。根据每个引物的合成报告单计算出配制10umol/L浓度的寡核苷酸需要加入水的体积，加入去离子无菌水，室温放置2min，反复振荡助溶，再离心将溶液收集到管底。 </p>
| |
| - | <p >每6条引物为一组，首末位引物取4ul，中间引物取1ul，加入新的EP管中，再补充灭菌去离子水至20ul，混匀备用。 </p>
| |
| - | <table align="center" >
| |
| - | <tr >
| |
| - | <td width="67" valign="center" ><p >组别 </p></td>
| |
| - | <td width="103" valign="center" ><p >引物编号 </p></td>
| |
| - | <td width="65" valign="center" ><p >吸取量 </p></td>
| |
| - | <td width="85" valign="center" ><p >灭菌去离子水添加量 </p></td>
| |
| - | <td width="74" valign="center" ><p >总体积 </p></td>
| |
| - | <td width="108" valign="top" ><p >每PCR（50ul体系）吸取量 </p></td>
| |
| - | <td width="108" valign="center" ><p >终浓度 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="67" valign="center" rowspan="6" ><p >A1 </p></td>
| |
| - | <td width="103" valign="center" ><p >A101 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="85" valign="center" rowspan="6" ><p >8 </p></td>
| |
| - | <td width="74" valign="center" rowspan="6" ><p >20 </p></td>
| |
| - | <td width="108" valign="center" rowspan="6" ><p >5ul </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A102 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="center" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A103 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A104 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A105 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A106 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="67" valign="center" rowspan="6" ><p >A2 </p></td>
| |
| - | <td width="103" valign="center" ><p >A107 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="85" valign="center" rowspan="6" ><p >8 </p></td>
| |
| - | <td width="74" valign="center" rowspan="6" ><p >20 </p></td>
| |
| - | <td width="108" valign="center" rowspan="6" ><p >5ul </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A108 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="center" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A109 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A110 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A111 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A112 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="67" valign="center" rowspan="6" ><p >A3 </p></td>
| |
| - | <td width="103" valign="center" ><p >A113 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="85" valign="center" rowspan="6" ><p >8 </p></td>
| |
| - | <td width="74" valign="center" rowspan="6" ><p >20 </p></td>
| |
| - | <td width="108" valign="center" rowspan="6" ><p >5ul </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A114 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="center" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A201 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A202 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A203 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A204 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="67" valign="center" rowspan="6" ><p >A4 </p></td>
| |
| - | <td width="103" valign="center" ><p >A205 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="85" valign="center" rowspan="6" ><p >8 </p></td>
| |
| - | <td width="74" valign="center" rowspan="6" ><p >20 </p></td>
| |
| - | <td width="108" valign="center" rowspan="6" ><p >5ul </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A206 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="center" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A207 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A208 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A209 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A210 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="67" valign="center" rowspan="4" ><p >A5 </p></td>
| |
| - | <td width="103" valign="center" ><p >A211 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="85" valign="center" rowspan="4" ><p >10 </p></td>
| |
| - | <td width="74" valign="center" rowspan="4" ><p >20 </p></td>
| |
| - | <td width="108" valign="center" rowspan="4" ><p >5ul </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A212 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="center" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A213 </p></td>
| |
| - | <td width="65" valign="center" ><p >1 </p></td>
| |
| - | <td width="108" valign="top" ><p >50nM </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="103" valign="center" ><p >A214 </p></td>
| |
| - | <td width="65" valign="center" ><p >4 </p></td>
| |
| - | <td width="108" valign="center" ><p >200nM </p></td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - |
| |
| - | <h3 >3、DA-PCR </h3>
| |
| - | <p >化学合成的单链寡核苷酸，每6条分为一组，通过DA-PCR拼接成为较长的双链中间片段（Block）。DA-PCR的反应体系组成为： </p>
| |
| - | <p > Mixed primer solutions 5μl </p>
| |
| - | <p > Pfu DNA Polymerase 2.5U 0.5μl </p>
| |
| - | <p > dNTP 4μl </p>
| |
| - | <p > 10×Pfu buffer（Mg2+） 5μl </p>
| |
| - | <p > 灭菌超纯水 补齐至 50μl </p>
| |
| - | <p >DA-PCR的反应程序为： </p>
| |
| - |
| |
| - | <p > 94℃ 2min </p>
| |
| - | <p > 94℃ 30S </p>
| |
| - | <p > 47℃ 30S 25个循环 </p>
| |
| - | <p > 72℃ 1min </p>
| |
| - | <p > 72℃ 10min </p>
| |
| - | <p > 4℃ 保存 </p>
| |
| - | <p > End </p>
| |
| - | <p >DA-PCR拼接反应产物检测采用2％的琼脂糖凝胶电泳，其中 </p>
| |
| - | <p >DA-PCR产物 5μl </p>
| |
| - | <p >10×Loading Buffer 0.6μl 混合均匀后点样 </p>
| |
| - | <p >100bp Marker 5μl 直接点样 </p>
| |
| - | <p >75V电泳1h。 </p>
| |
| - | <p >	 </p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/8/84/Simonsong-result-1.png" ></p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <p >图1 DA-PCR拼接结果 </p>
| |
| - | <p >结果说明： </p>
| |
| - | <p >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。 </p>
| |
| - | <p >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）。 </p>
| |
| - | <p > </p>
| |
| - | <h3 >4、OE-PCR </h3>
| |
| - | <p >以DA-PCR拼接出来的的5条双链中间片段（Block）为模版，进一步利用OE-PCR进行连接，OE-PCR的反应体系组成为： </p>
| |
| - | <p > Block1 1μl </p>
| |
| - | <p > Block2 1μl </p>
| |
| - | <p > Block3 1μl </p>
| |
| - | <p > Block4 1μl </p>
| |
| - | <p > Block5 1μl </p>
| |
| - | <p > A1 1μl </p>
| |
| - | <p > A4 1μl </p>
| |
| - | <p > Pfu DNA Polymerase 2.5U 0.5μl </p>
| |
| - | <p > dNTP 4μl </p>
| |
| - | <p > 10×Pfu buffer（Mg2+） 5μl </p>
| |
| - | <p ><img width="349" height="2" src="3_wpsE664.tmp.png" > 灭菌超纯水 补齐至 50μl </p>
| |
| - | <p >OE-PCR反应程序为： </p>
| |
| - | <p > 94℃ 2min </p>
| |
| - | <p ><img width="13" height="64" src="3_wpsE675.tmp.png" > 94℃ 30S </p>
| |
| - | <p > 47℃ 30S 20个循环 </p>
| |
| - | <p > 72℃ 2min </p>
| |
| - | <p > 72℃ 10min </p>
| |
| - | <p ><img width="349" height="2" src="3_wpsE686.tmp.png" > 4℃ 保存 </p>
| |
| - | <p > End </p>
| |
| - | <p >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 </p>
| |
| - | <p ><img width="13" height="34" src="3_wpsE687.tmp.png" >OE-PCR产物 5μl </p>
| |
| - | <p >10×Loading Buffer 0.6μl 混合均匀后点样 </p>
| |
| - | <p ><img width="349" height="2" src="3_wpsE688.tmp.png" >100bp Marker 5μl 直接点样 </p>
| |
| - | <p >80V电泳1h。 </p>
| |
| - | <p > </p>
| |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/2/24/Simonsong-result-2.png" ></p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <p >图2 OE-PCR拼接结果 </p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <p >结果说明： </p>
| |
| - | <p >1、L1、L2、L3都能得到5条中间产物拼接到一起的mlrA全基因。 </p>
| |
| - | <p >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。 </p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <h3 >5、亚克隆载体构建与测序 </h3>
| |
| - | <p >合成的mlrA基因与pSB1C3载体经过EcoRⅠ和PstⅠ于37℃双酶切3h后，16℃连接过夜，转化到E.coli JM109感受态细胞中，经蓝白筛选挑取白色菌落。 </p>
| |
| - | <p >EcoRⅠ和PstⅠ双酶切反应体系组成为： </p>
| |
| - | <p > Gene or plasmid 10μl </p>
| |
| - | <p > EcoRⅠ 0.4μl </p>
| |
| - | <p > PstⅠ 0.2μl </p>
| |
| - | <p > 10×NEBuffer 3.1 2μl </p>
| |
| - | <p > 灭菌超纯水 补齐至 20μl </p>
| |
| - | <p >连接反应体系组成为： </p>
| |
| - | <p > mlrA Gene 10μl </p>
| |
| - | <p > pSB1C3 3μl </p>
| |
| - | <p > T4 ligase 0.5μl </p>
| |
| - | <p > 10×T4 ligase Buffer 2μl </p>
| |
| - | <p > 灭菌超纯水 补齐至 20μl </p>
| |
| - | <p >E.coli JM109感受态细胞制备流程： </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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℃。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >热激转化过程： </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >平板涂布及培养过程： </p>
| |
| - | <p >(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。 </p>
| |
| - | <p >(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； </p>
| |
| - | <p >实验连接组：连接产物，氯霉素板 </p>
| |
| - | <p >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 </p>
| |
| - | <p >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。 </p>
| |
| - | <p >空菌组：感受态细胞，无氯霉素板 </p>
| |
| - | <p >序列测定 </p>
| |
| - | <p >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）。 </p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/0/0d/Simonsong-result-3.png" > </p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <p >图3 重组载体pSB1C3-A </p>
| |
| - | <p >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。 </p>
| |
| - |
| |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/6/63/Simonsong-result-4.png" ></p>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <p > 图4 重组载体pSB1C3-A的PCR鉴定 </p>
| |
| - | <p >结果说明： </p>
| |
| - | <p >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 </p>
| |
| - | <p >2、M是100bp Ladder Marker </p>
| |
| - | <p >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。 </p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <p ><img src="https://static.igem.org/mediawiki/2014/5/5a/Simonsong-result-5.png" ><img src="https://static.igem.org/mediawiki/2014/0/0b/Simonsong-result-6.png" > </p>
| |
| - | <p >图5 测序结果与设计序列比对 </p>
| |
| - | <p > </p>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <h3 ><img width="155" height="58" src="6_wps9D1F.tmp.png" >6、引物合成 </h3>
| |
| - | <table >
| |
| - | <tr >
| |
| - | <td width="1038" valign="center" rowspan="3" ><p > </p>
| |
| - | <table >
| |
| - | <tr >
| |
| - | <td width="1023" valign="center" rowspan="2" ><p >吉林省库美生物科技有限公司引物合成订购单 </p></td>
| |
| - | </tr>
| |
| - | <tr ></tr>
| |
| - | </table>
| |
| - | </table>
| |
| - | <p > </p>
| |
| - | <table >
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >引物编号 </p></td>
| |
| - | <td width="456" valign="center" ><p >序列，(5' to 3') </p></td>
| |
| - | <td width="57" valign="center" ><p >碱基数（bp） </p></td>
| |
| - | <td width="57" valign="center" ><p >合成总量(OD) </p></td>
| |
| - | <td width="47" valign="center" ><p >分装管数 </p></td>
| |
| - | <td width="56" valign="center" ><p >纯化方式 </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A101 </p></td>
| |
| - | <td width="456" valign="center" ><p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCT </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A102 </p></td>
| |
| - | <td width="456" valign="center" ><p >CTCGTAAAGCATGAGCAGCTAAAGCAATTAAAATAGCTAAAACATAAAAACATAATA </p></td>
| |
| - | <td width="57" valign="center" ><p >57 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A103 </p></td>
| |
| - | <td width="456" valign="center" ><p >AGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTT </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A104 </p></td>
| |
| - | <td width="456" valign="center" ><p >AGCAGTAATAATATTTAAATGAGCATGAGTTTCTTGTAACATTTTAAACATTGGATCTAA </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A105 </p></td>
| |
| - | <td width="456" valign="center" ><p >CTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTT </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A106 </p></td>
| |
| - | <td width="456" valign="center" ><p >CAGCAAACATTGGAGCAGCTGGAAATAATAATAAAGTATAAGCTCCTGGATATTCA </p></td>
| |
| - | <td width="57" valign="center" ><p >56 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A107 </p></td>
| |
| - | <td width="456" valign="center" ><p >GCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTT </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A108 </p></td>
| |
| - | <td width="456" valign="center" ><p >TCGCCATGGAGCACATCGTGATAATAATTCTCGAAATCCAGCTTGTCCATATCCAA </p></td>
| |
| - | <td width="57" valign="center" ><p >56 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A109 </p></td>
| |
| - | <td width="456" valign="center" ><p >ACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGT </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A110 </p></td>
| |
| - | <td width="456" valign="center" ><p >CATAATTCCAGTTAAAGCAAAAAAAGCTAAAAAACAAACAGCAATAACAGTAACTCCTTG </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A111 </p></td>
| |
| - | <td width="456" valign="center" ><p >TTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTT </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A112 </p></td>
| |
| - | <td width="456" valign="center" ><p >CAACTGGATCTGATCCATATCGTAAAAAAGTTCGATCTAAAGTTCCTGGTGGAGCATATA </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A113 </p></td>
| |
| - | <td width="456" valign="center" ><p >CGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCA </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A114 </p></td>
| |
| - | <td width="456" valign="center" ><p >ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAGCAGCT </p></td>
| |
| - | <td width="57" valign="center" ><p >44 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A201 </p></td>
| |
| - | <td width="456" valign="center" ><p >ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCC </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A202 </p></td>
| |
| - | <td width="456" valign="center" ><p >CACCACATAATTCCTAAAATAACAGCAGCAGTTAATGGATCAAATTTTTTTAATAATTGT </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A203 </p></td>
| |
| - | <td width="456" valign="center" ><p >TGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATT </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A204 </p></td>
| |
| - | <td width="456" valign="center" ><p >AACTGACCAAGCAGCTCCTGGAGCTCCTGAAAATAAAGTTGGTAAATCTCGTGGT </p></td>
| |
| - | <td width="57" valign="center" ><p >55 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A205 </p></td>
| |
| - | <td width="456" valign="center" ><p >AGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTC </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A206 </p></td>
| |
| - | <td width="456" valign="center" ><p >CCTAATTTATTACAAACAAAAACAGCAATAATAGTTGAAGCAATAAATCCTGGAGTAATA </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A207 </p></td>
| |
| - | <td width="456" valign="center" ><p >TGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGC </p></td>
| |
| - | <td width="57" valign="center" ><p >57 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A208 </p></td>
| |
| - | <td width="456" valign="center" ><p >CATTCAGCAGTAACATTAACTCCTAATTCATTATGAATAGCATGAGTTAAAACTCCTCCC </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A209 </p></td>
| |
| - | <td width="456" valign="center" ><p >AGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGA </p></td>
| |
| - | <td width="57" valign="center" ><p >55 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A210 </p></td>
| |
| - | <td width="456" valign="center" ><p >ACTAATCCAATAGCAACAGCAAATTCAATTAAATCCCATGGTCGCCATCCTAATCCAGCA </p></td>
| |
| - | <td width="57" valign="center" ><p >60 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A211 </p></td>
| |
| - | <td width="456" valign="center" ><p >ATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTC </p></td>
| |
| - | <td width="57" valign="center" ><p >57 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A212 </p></td>
| |
| - | <td width="456" valign="center" ><p >GTGGAACATTTCCCCAAGCTAATCGAGCATTATCTGGTGAAGCAGCTCCTAATGATCGT </p></td>
| |
| - | <td width="57" valign="center" ><p >59 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A213 </p></td>
| |
| - | <td width="456" valign="center" ><p >TTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGC </p></td>
| |
| - | <td width="57" valign="center" ><p >59 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A214 </p></td>
| |
| - | <td width="456" valign="center" ><p >AAGACGTCCTATTAAGCATTAGCTCCTGATTTATCTCCAACTC </p></td>
| |
| - | <td width="57" valign="center" ><p >43 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A1 </p></td>
| |
| - | <td width="456" valign="center" ><p >CCGGAATTCATGCGAGAATTTGTTCG </p></td>
| |
| - | <td width="57" valign="center" ><p >26 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A2 </p></td>
| |
| - | <td width="456" valign="center" ><p >ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAG </p></td>
| |
| - | <td width="57" valign="center" ><p >39 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A3 </p></td>
| |
| - | <td width="456" valign="center" ><p >ATTAGAAGAATTAGGATGGCGAGGATTTGC </p></td>
| |
| - | <td width="57" valign="center" ><p >30 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A4 </p></td>
| |
| - | <td width="456" valign="center" ><p >AAGACGTCCTATTAAGCATTAGCTCCTG </p></td>
| |
| - | <td width="57" valign="center" ><p >28 </p></td>
| |
| - | <td width="57" valign="center" ><p >1 </p></td>
| |
| - | <td width="47" valign="center" ><p >1 </p></td>
| |
| - | <td width="56" valign="center" ><p >PAGE </p></td>
| |
| - | </tr>
| |
| - | <tr >
| |
| - | <td width="50" valign="center" ><p >A5 </p></td>
| |
| - | <td width="456" valign="center" ><p >AACTGCAGCTATTAAGCATTAGCTCCTG </p></td>
| |
| - | <td width="57" valign="center" ><p >28 </p></td>
| |
| - | <td width="57" valign="center" ><p > </p></td>
| |
| - | <td width="47" valign="center" ><p > </p></td>
| |
| - | <td width="56" valign="center" ><p > </p></td>
| |
| - | </tr></div>
| |
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