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Team:Jilin China/RESULT
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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/ | + | <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"> | + | <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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<li><a style="margin-top:20px;" class="hide" href="https://2014.igem.org/Team:Jilin_China/Acknowledgement">ACKNOWLEDGEMENT</a></li> | <li><a style="margin-top:20px;" class="hide" href="https://2014.igem.org/Team:Jilin_China/Acknowledgement">ACKNOWLEDGEMENT</a></li> | ||
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| + | <title>NOTEBOOK</title> | ||
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| + | <!--内容--> | ||
| + | <div id="coop"> | ||
| + | <center><h2>Synthesis of gene mlrA</h2></center> | ||
| + | <h3>The experimental scheme</h3> | ||
| + | <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 > | + | <h3 >2、The selection of codons</h3> |
| - | < | + | <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 >GUA 12.8( 8921) GCA 22.3( 15521) GAA 56.1( 39035) GGA 24.5( 17046) </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 >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> | ||
| - | < | + | <h4>The codon table used</h4> |
| - | + | ||
| - | + | ||
<p >* TAA </p> | <p >* TAA </p> | ||
<p >A GCT </p> | <p >A GCT </p> | ||
| Line 247: | Line 262: | ||
<p >W TGG </p> | <p >W TGG </p> | ||
<p >Y TAT </p> | <p >Y TAT </p> | ||
| - | + | <h3>3、The inverse translation sequences and the selection of restriction enzymes</h3> | |
| - | <h3 > | + | |
<p >EcoR I </p> | <p >EcoR I </p> | ||
<p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGCTGCAGTT </p> | <p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGCTGCAGTT </p> | ||
| - | + | <h3 >4、The sequence analysis</h3> | |
| - | + | ||
| - | <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> | ||
| Line 274: | Line 286: | ||
<p >NlaIV (821) </p> | <p >NlaIV (821) </p> | ||
<p >Hpy166II (901) </p> | <p >Hpy166II (901) </p> | ||
| - | <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> | ||
| Line 303: | Line 315: | ||
                                    TGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCAC            TGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAA                 ATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCC            CCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTAC            ACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCA            TGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTG               CTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA<br> |                                     TGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCAC            TGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAA                 ATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCC            CCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTAC            ACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCA            TGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTG               CTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA<br> | ||
TAATCTTCTTAATCCTACCGCTCCTAAACGAAATGGTGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCACCCGAACCGTAAATGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAATAACAATTTGTTAATCAATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCCTCCTAGTTACACCCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTACCCGAGGTTGACAACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCAAAATTAAACACCTGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTGGTTTTAATGGTCCTCCTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA </p> | TAATCTTCTTAATCCTACCGCTCCTAAACGAAATGGTGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCACCCGAACCGTAAATGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAATAACAATTTGTTAATCAATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCCTCCTAGTTACACCCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTACCCGAGGTTGACAACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCAAAATTAAACACCTGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTGGTTTTAATGGTCCTCCTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA </p> | ||
| - | + | <h3>The experimental procedure and analysis</h3> | |
| - | + | <h3 >1、Dissolving of primers</h3> | |
| - | < | + | <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> |
| - | <h3 > | + | |
| - | <p > | + | |
<table align="center" > | <table align="center" > | ||
<tr > | <tr > | ||
| - | <td width="47" valign="center" ><p > | + | <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> | ||
| Line 548: | Line 558: | ||
</tr> | </tr> | ||
</table> | </table> | ||
| - | + | <h3 id="coop">2、Mixing of primers</h3> | |
| - | <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 > | + | <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 > | + | <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 > | + | <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" | + | <td width="103"><p align="center"><strong>Number of primer</strong></p></td> |
| - | <td width="103" | + | <td width="65"><p align="center"><strong>Uptake</strong></p></td> |
| - | <td width="65" | + | <td width="85"><p align="center"><strong>The volume of sterile deionized water</strong></p></td> |
| - | <td width="85" | + | <td width="75"><p align="center"><strong>The total volume</strong></p></td> |
| - | <td width=" | + | <td width="108" valign="top"><p align="center"><strong>Each PCR (50ul system) uptake</strong></p></td> |
| - | <td width="108" valign="top" ><p > | + | <td width="108"><p align="center"><strong>Final concentration</strong></p></td> |
| - | <td width="108" | + | |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="6"><p align="center">A1</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A101</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" | + | <td width="75" rowspan="6"><p align="center"><a name="_GoBack"></a>20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="6"><p align="center">5ul</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A102</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A103</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A104</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A105</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A106</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="6"><p align="center">A2</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A107</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" | + | <td width="75" rowspan="6"><p align="center">20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="6"><p align="center">5ul </p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A108</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A109</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A110</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A111</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A112</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="6"><p align="center">A3</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A113</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" | + | <td width="75" rowspan="6"><p align="center">20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="6"><p align="center">5ul </p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A114</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A201</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A202</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A203</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A204</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="6"><p align="center">A4</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A205</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="6"><p align="center">8</p></td> |
| - | <td width=" | + | <td width="75" rowspan="6"><p align="center">20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="6"><p align="center">5ul </p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A206</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A207</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A208</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A209</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A210</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="67 | + | <td width="67" rowspan="4"><p align="center">A5</p></td> |
| - | <td width="103" | + | <td width="103"><p align="left">A211</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="85 | + | <td width="85" rowspan="4"><p align="center">10</p></td> |
| - | <td width=" | + | <td width="75" rowspan="4"><p align="center">20</p></td> |
| - | <td width="108 | + | <td width="108" rowspan="4"><p align="center">5ul </p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A212</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">50nM</p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A213</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">1</p></td> |
| - | <td width="108" valign="top" ><p >50nM </p></td> | + | <td width="108" valign="top"><p align="center">50nM </p></td> |
</tr> | </tr> | ||
| - | <tr > | + | <tr> |
| - | <td width="103" | + | <td width="103"><p align="left">A214</p></td> |
| - | <td width="65" | + | <td width="65"><p align="center">4</p></td> |
| - | <td width="108" | + | <td width="108"><p align="center">200nM</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
| - | <h3 >3、DA-PCR </h3> | + | <h3 id="coop">3、DA-PCR </h3> |
| - | <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 > | + | <p id="coop">Procedure of DA-PCR： <br> |
| - | < | + | Mixed primer solutions 5μl<br> |
| - | < | + | Pfu DNA Polymerase 2.5U 0.5μl <br> |
| - | < | + | dNTP 4μl<br> |
| - | < | + | 10×Pfu buffer（Mg2+） 5μl<br> |
| - | < | + | 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> | ||
| - | < | + | <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 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/ | + | 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 > | + | <p id="coop">Illustration of the outcome:</p> |
| - | < | + | <ol id="coop"> |
| - | < | + | <li>L1, L2, L3 could get mlrA genome which were made by five single-stranded oligonucleotides splicedtogether.</li> |
| - | < | + | <li>Wide strip of the tape at 100bp was caused by excessive amount of amplification primers A1 and A4.</li> |
| - | + | </ol> | |
| - | <h3 > | + | <h3 id="coop">5、Constructing and sequencing of subcoloning vector</h3> |
| - | <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> |
| - | < | + | 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> |
| - | < | + | Gene or plasmid 10μl<br> |
| - | < | + | <em>Eco</em>RⅠ 0.4μl<br> |
| - | < | + | <em>Pst</em>Ⅰ 0.2μl<br> |
| - | < | + | 10×NEBuffer 3.1 2μl<br> |
| - | < | + | sterilizing ulturapure water top up to 20μl<br> |
| - | < | + | <strong>Constitute of linking reaction system</strong><strong>：</strong><strong> </strong><br> |
| - | < | + | mlrA Gene 10μl<br> |
| - | < | + | pSB1C3 3μl<br> |
| - | < | + | T4 ligase 0.5μl<br> |
| - | < | + | 10×T4 ligase Buffer 2μl<br> |
| - | < | + | sterilizing ulturapure water top up to 20μl<br> |
| - | < | + | Preparation process of competent cell <strong><em>E.coli</em>BL21</strong><strong>：</strong><strong> </strong><br> |
| - | < | + | (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> |
| - | < | + | (2) In the aseptic condition, transfer 30mLbacteria fluid to the centrifugal tube in the ice 10 min to cooling the culture to 0℃.<br> |
| - | < | + | (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> |
| - | < | + | (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> |
| - | < | + | <strong>Heal shock transformation process</strong><strong>：</strong><strong> </strong><br> |
| - | < | + | (1) Thawing freshly prepared or -80℃ competentcellBL21suspension.<br> |
| - | < | + | (2) Add constructed recombinant cloning vector plasmidspSB1C3-A(no more than 50ng, 10μl), shake gently, and then in the ice 30min.<br> |
| - | < | + | (3) Put EP tube in 42℃ water bath90s，and then transfer it to ice water bath to cool cells1-2 min.<br> |
| - | < | + | (4) add 800μL LB medium to EP tube，transfer it to 37℃ table，150r/min，to recovery strain 45 min.<br> |
| - | < | + | Spread plate and Culture<br> |
| - | < | + | (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> |
| - | < | + | (2) Wait until the solution in culture is full absorbed by cell, then invert the plate and culture it in 37℃ overnight.<br> |
| - | < | + | 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 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> | ||
| - | |||
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| + | <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> | ||
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| - | + | <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> | |
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Latest revision as of 03:59, 18 October 2014
Welcome!
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2、Mixing of primersOligonucleotides synthesized in powder form need to be centrifuged for 1 min at 10000rpm to assemble powders to the bottom of the EP tube.And be careful to open the cap. Make sure that the synthesis report is consistent to the OD number on the primer label,and check the number of the dispensing tube before the dissolution of oligonucleotides.The volume of water added to make 10umol/L oligonucleotides’ solution is calculated according to the synthetic single report of each primer. Add sterile deionized water and keep it at room temperature for 2min.And reverse the tube to accelerate the progress of solubilization. Finally, collect substances at the bottom of the tube after centrifugation. Every six primers are combined to one group.Add 4ulbeginning fiprimer, 1ulintermediate primer and 4ul end primerto a new EP tube, then add sterile deionized water to 20ul, mix them up as spare.
3、DA-PCREvery six primers which are single-stranded oligonucleotidesare chemically synthesized were combined to one group. Then they were going to be made as longer double-stranded fragments of intermediate (Block) by DA-PCR Procedure of DA-PCR：
Figure1 DA-PCR splicing results Illustration of the outcome: 4、OE-PCRTake the 5 double strand intermediate fragment (Block) spliced by DA-PCR as templates, blocks were further connected by OR-PCR, OE-PCR in a reaction system consisting of: Block1 1μl
Illustration of the outcome:
5、Constructing and sequencing of subcoloning vectorAfter double enzyme digestion reaction at 37℃ for 3h by EcoRⅠand PstⅠ, MlrA gene and pSB1C3 vector linked together at 16℃ overnight. And then transformed to E.coli JM109 competent cell, and select the desirable colony by using “blue-white selection” method.
Fig3.Reconstructed vector pSB1C3-A 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.
Fig 4. The identification of reconstructed vector pSB1C3-A Results: 1.L1 and L2 is 1300bp and it confirms that the reconstructed vector has mlrA on it. 2.M is the 100bp Ladde Marker. Sequencing result shows that the sequence of synthetic mlrA gene is exactly same as sequence of designed sequence.
Fig5.Comparison of result of sequencing and designed sequence
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