Difference between revisions of "Team:Hong Kong HKU/Results"

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{{Hong_Kong_HKU}}
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<h2> Project Results</h2>
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<p>Here you can describe the results of your project and your future plans. </p>
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<h5>What should this page contain?</h5>
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        h2
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<ul>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project </li>
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<li> Considerations for replicating the experiments </li>
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<h4> Project Achievements </h4>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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      <ul>
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        <li id="line"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU">HOME</a></li>
 +
        <li id="line"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Team">TEAM</a></li>
 +
        <li id="line"><a href = "#">PROJECT</a>
 +
          <ul style="margin-left: 18%;">
 +
            <li style = "margin-top: 3px"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Description">Description</a></li>
 +
            <li style = "margin-top: 3px"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Experiments">Experiments</a></li>
 +
            <li style = "margin-top: 3px"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Results">Results</a></li>
 +
            <li style = "margin-top: 3px"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Design">Design</a></li>
 +
          </ul>
 +
        </li>
 +
        <li id="line"><a href = "#">PARTS</a>
 +
          <ul style="margin-left:26.6%;">
 +
            <li style = "margin-top: 3px"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Parts">Team Parts</a></li>
 +
            <li style = "margin-top: 3px"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Basic_Part">Basic Parts</a></li>
 +
            <li style = "margin-top: 3px"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Composite_Part">Composite <br> Parts</a></li>
 +
          </ul>
 +
        </li>
 +
        <li id="line"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Modeling">MODELING</a></li>
 +
                                <li id="line"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Notebook">NOTEBOOK</a></li>
 +
        <li id="line"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Attributions">ATTRIBUTIONS</a></li>
 +
        <li id="line"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Practices">HUMANPRACTICES</a></li>
 +
        <li id="line"><a href = "https://2015.igem.org/Team:Hong_Kong_HKU/Safety">SAFETY</a></li>
 +
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 +
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 +
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 +
 
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 +
                        <h3>RESULT</h3>
 +
 +
<h2>Design and Order gBlock</h2>
 +
 +
<p>After the construction and optimization of our killing switch system that aims to work in E. coli BL21(DE3), we distribute them into 5 gBlock fragments, a synthetic double-stranded DNA product provided by IDT company. This strategy is chosen since our design included several new parts such as Cas9 and guiding RNA that target specific sequences. The whole construct also involved in numerous minor changes, thus traditional cloning and assembly of available biobricks cannot meet our needs.</p>
 +
</div>
 +
 +
<img id = "circuit_img" src = "https://static.igem.org/mediawiki/2015/a/a6/Gblock_khu.jpeg"/>
 +
 +
<div id="contentcontainer">
 +
<h6 style= "text-align: center;">Fig 1. Span of gBlock corresponding to the killing switch construct. For easy understanding, the length of DNA fragments in the figure are not in actual proportion. </h6>
 +
<table width="100%"  cellspacing="0" height="20px" border = "0"> </table>
 +
 +
<p>These gBlocks are DNA fragments synthesized according to our order, and can be later assembled by Hi-Fi assembly. Fig. 1 shows the span of the gBlock fragments and their length in base pair. gBlock 1 initiates with prefix and gblock 5 ends with suffix, thus after assembly with pSB1C3 backbone, the whole construct forms a circular plasmid and can be transformed into targeted organisms.</p>
 +
 +
 +
 +
 +
<h2>Design and Order gBlock Primer</h2>
 +
<table align="center">
 +
  <tr>
 +
    <td>
 +
  <img src = "https://static.igem.org/mediawiki/2015/4/48/Result_fig2.jpeg"/>
 +
</td>
 +
</tr>
 +
</table>
 +
<h6 style= "text-align: center;">Fig. 2  Relative position of primers and gBlocks</h6>
 +
<table width="100%"  cellspacing="0" height="20px" border = "0"> </table>
 +
 +
<p>While ordering the gBlock DNAs, we also designed and ordered the 10 forward and reverse primers that matched the 5 gBlocks respectively in order to amplify them through PCR. Fig. 2 illustrates the relative positions of gBlocks and primers in a circular plasmid. Since the forward/ reverse primers have overlapping regions on the preceding/ following gBlocks, after amplification all fragments would have overlapping sequences that enable Hi-Fi assembly. The sequences and working temperatures of each primers are provided in the table below. All primers were designed to have around 40 base pairs to provide enough specificity for the annealing DNA. Yet the arrangement exceeded the optimal length of primer design (which should be 18-24 bp) and the primers might have a higher chance to generate self-dimers or form secondary structure.</p>
 +
 +
 +
<table id="t01" style="width:100%">
 +
  <tr>
 +
    <th>Overlaps</th>
 +
    <th>Oligo (Uppercase = gene-specific primer) 5'->3'</th>
 +
    <th>Anneals</th>
 +
    <th>F/R</th>
 +
    <th>3' Tm</th>
 +
    <th>3' Ta</th>
 +
  </tr>
 +
  <tr>
 +
    <td>gBlock 4</td>
 +
    <td><font color="#ea662d">tgggcctttctgcgtttataTACTAGAGTTTACGGCTAG</font></td>
 +
    <td>gBlock 5</td>
 +
    <td>Fwd</td>
 +
    <td>55.1°C</td>
 +
    <td>58.1°C</td>
 +
  </tr>
 +
  <tr>
 +
    <td>pSBCc3</td>
 +
    <td><font color="#454545">tgcagcggccgctactagtaAAAAAAAGCACCGACTCG</font></td>
 +
    <td>gBlock 5</td>
 +
    <td>Rev</td>
 +
    <td>58.3°C</td>
 +
    <td>58.1°C</td>
 +
  </tr>
 +
    <tr>
 +
    <td>pSB1C3</td>
 +
    <td><font color="#454545">attcgcggccgcttctagagTTATGACAACTTGACGGC</font></td>
 +
    <td>gBlock 1</td>
 +
    <td>Fwd</td>
 +
    <td>57.1°C</td>
 +
    <td>58.4°C</td>
 +
  </tr>
 +
    <tr>
 +
    <td>gBlock 2</td>
 +
    <td><font color="#eeb400">cgtgcaaataatcaatgtctCTAGTAATAGCAAAGTGTGAC</font></td>
 +
    <td>gBlock 1</td>
 +
    <td>Rev</td>
 +
    <td>55.4°C</td>
 +
    <td>58.4°C</td>
 +
  </tr>
 +
    <tr>
 +
    <td>gBlock 3</td>
 +
    <td><font color="#0d6a98">aactgtcaaagttgttgatgAATTAGTAAAAGTGATGGGTCG</font></td>
 +
    <td>gBlock 4</td>
 +
    <td>Fwd</td>
 +
    <td>57.7°C</td>
 +
    <td>59.9°C</td>
 +
  </tr>
 +
    <tr>
 +
    <td>gBlock 5</td>
 +
    <td><font color="#4f854d">gctagccgtaaactctagtaTATAAACGCAGAAAGGCC</font></td>
 +
    <td>gBlock 4</td>
 +
    <td>Rev</td>
 +
    <td>56.7°C</td>
 +
    <td>59.9°C</td>
 +
  </tr>
 +
    <tr>
 +
    <td>gBlock 2</td>
 +
    <td><font color="#eeb400">caatctgatcgcattgtcgcTGGGTCTGACCCCTAACT</font></td>
 +
    <td>gBlock 3</td>
 +
    <td>Fwd</td>
 +
    <td>62.5°C</td>
 +
    <td>60.9°C</td>
 +
  </tr>
 +
    <tr>
 +
    <td>gBlock 4</td>
 +
    <td><font color="#ea662d">acccatcacttttactaattCATCAACAACTTTGACAGTTTG</font></td>
 +
    <td>gBlock 3</td>
 +
    <td>Rev</td>
 +
    <td>57.9°C</td>
 +
    <td>60.9°C</td>
 +
  </tr>
 +
    <tr>
 +
    <td>gBlock 1</td>
 +
    <td><font color="#d40909">tcacactttgctattactagAGACATTGATTATTTGCACGG</font></td>
 +
    <td>gBlock 2</td>
 +
    <td>Fwd</td>
 +
    <td>58.5°C</td>
 +
    <td>61.5°C</td>
 +
  </tr>
 +
    <tr>
 +
    <td>gBlock 3</td>
 +
    <td><font color="#0d6a98">aaagttaggggtcagacccaGCGACAATGCGATCAGATTG</font></td>
 +
    <td>gBlock 2</td>
 +
    <td>Rev</td>
 +
    <td>62.3°C</td>
 +
    <td>61.5°C</td>
 +
  </tr>
 +
    <tr style="background-color=#aeaeae;">
 +
    <td>---</td>
 +
    <td>CTCTAGAAGCGGCCGCGA</td>
 +
    <td>pSB1C3</td>
 +
    <td>Rev</td>
 +
    <td>68.1°C</td>
 +
    <td>67.5°C</td>
 +
  </tr>
 +
    <tr style="background-color=#aeaeae;">
 +
    <td>---</td>
 +
    <td>TACTAGTAGCGGCCGCTG</td>
 +
    <td>pSB1C3</td>
 +
    <td>Fwd</td>
 +
    <td>64.5°C</td>
 +
    <td>67.5°C</td>
 +
  </tr>
 +
 +
</table>
 +
 +
<h6 style= "text-align: center;"> Table 1. Sequences of the 10 ordered forward and reverse primers. The lowercase letters correspond to the overlapping sequences and the capital letters represent the annealing sequences of the adjacent gBlock fragments. Tm stands for melting temperature, and Ta stands for annealing temperature. </h6>
 +
<table width="100%"  cellspacing="0" height="20px" border = "0"> </table>
 +
 +
 +
 +
 +
 +
 +
 +
 +
<h6> 4. If Taq polymerase is used instead of Q5, assemble all reaction components as followed on ice. Gently mix all the components before use. Collect all liquid to the bottom of the tube by a quick spin if necessary. </h6>
 
<ul>
 
<ul>
−
<li>A list of linked bullet points of the successful results during your project</li>
+
<table style="width:80%">
−
<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
+
  <tr>
 +
    <th>COMPONENT</th>
 +
    <th>50 uL REACTION</th>
 +
  </tr>
 +
  <tr>
 +
    <td>Taq buffer</td>
 +
    <td>5 uL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>dNTPs</td>
 +
    <td>1 uL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>10 uM Forward Primer</td>
 +
    <td>1 uL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>10 uM Reverse Primer</td>
 +
    <td>1 uL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Taq polymerase</td>
 +
    <td>0.5 uL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Template gBlock DNA</td>
 +
    <td>2 uL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>PCR water</td>
 +
    <td>39.5 uL</td>
 +
  </tr>
 +
 
 +
</table>
 
</ul>
 
</ul>
 +
<h6> 5. Transfer PCR tubes to a PCR machine and begin thermocycling.</h6>
 +
<ul>
 +
<table style="width:80%">
 +
  <tr>
 +
    <th>STEP</th>
 +
    <th>TEMPERATURE</th>
 +
    <th>TIME</th>
 +
  </tr>
 +
  <tr>
 +
    <td>Initial Denaturation</td>
 +
    <td>98 degree Celsius</td>
 +
    <td>30 second </td>
 +
  </tr>
 +
  <tr>
 +
    <td>30 cycles</td>
 +
    <td>98 degree Celsius</td>
 +
    <td>10 seconds</td>
 +
  </tr>
 +
  <tr>
 +
    <td> </td>
 +
    <td>50-72 degree Celsius</td>
 +
    <td>30 seconds</td>
 +
  </tr>
 +
  <tr>
 +
    <td> </td>
 +
    <td>72 degree Celsius</td>
 +
    <td>1.5 minutes </td>
 +
  </tr>
 +
  <tr>
 +
    <td>Final Extension</td>
 +
    <td>72 degree Celsius</td>
 +
    <td>2.5 minutes</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Hold</td>
 +
    <td>4 degree Celsius</td>
 +
    <td> </td>
 +
  </tr>
  
  
 +
</table>
 +
</ul>
  
−
<h4>Inspiration</h4>
+
</ul>
−
<p>See how other teams presented their results.</p>
+
<h2>Gel Extraction</h2>
 
<ul>
 
<ul>
−
<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
+
<p>-  QIAquick Gel Extraction Kit</p>
−
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
+
<ul>
−
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
+
<h6> 1. Cut the specific DNA fragments from the agarose gel with a scalpel. </h6>
 +
<h6> 2. Weigh the gel slice in an Eppendorf tube. Add 3 volumes buffer QG to 1 volume gel (100 mg gel to 100 uL). </h6>
 +
<h6> 3. Incubate the tube at 50 degree Celsius for 10 minutes. Vortex the tube every 2-3 minutes to help dissolve gel. The colour of the mixture is yellow.</h6>
 +
<h6> 4. Add 1 gel volume isopropanol to the sample and mix.</h6>
 +
<h6> 5. Place a QIAquick column in s provided 2-mL collection tube. Apply the sample to the QIAquick column and centrifuge for 1 minute. Discard the flow-through and place back the column back into the same tube (binding DNA).</h6>
 +
<h6> 6. Add 750 uL of buffer PE/EtOH to QIAquick column. Let the column stand for 4 minutes and centrifuge for 1 minute. Discard the flow-through and place back the QIAquick column back into the same tube (washing). Centrifuge the QIAquick column in the provided 2-mL collection tube for 1 minute to remove residual wash buffer. </h6>
 +
<h6> 7. Place the QIAquick column into a clean Eppendorf tube. </h6>
 +
<h6> 8. Add 30 uL of PCR water to the centre of the QIAquick membrane to elute DNA. Let the column stand for 1 minute and centrifuge for 1 minute. </h6>
 +
 
 +
</ul>
 
</ul>
 
</ul>
  
−
</div>
+
<h2>HiFi DNA Assembly and Transformation</h2>
 +
<ul>
 +
  <p>-  NEBuilder HiFi DNA Assembly Master Mix</p>
 +
  <p>-  Positive control provided: 2 overlapping dsDNA fragments for control assembly</p>
 +
<ul>
 +
<table style="width:80%">
 +
  <tr>
 +
    <td> </td>
 +
    <td>length (bp)</td>
 +
    <td>Conc. (ng/uL)</td>
 +
    <td>pmol needed</td>
 +
    <td>Mass (ng)</td>
 +
    <td>Vol. (uL)</td>
 +
  </tr>
 +
  <tr>
 +
    <td>gBlock 1</td>
 +
    <td>1160</td>
 +
    <td>19.5 </td>
 +
    <td>0.05</td>
 +
    <td>35.84</td>
 +
    <td>1.84</td>
 +
  </tr>
 +
  <tr>
 +
    <td>gBlock 2</td>
 +
    <td>1889</td>
 +
    <td>10 [stock]</td>
 +
    <td>0.05</td>
 +
    <td>58.37</td>
 +
    <td>5.84</td>
 +
  </tr>
 +
  <tr>
 +
    <td>gBlock 3</td>
 +
    <td>1540</td>
 +
    <td>23.8 </td>
 +
    <td>0.05</td>
 +
    <td>47.58</td>
 +
    <td>2.00</td>
 +
  </tr>
 +
  <tr>
 +
    <td>gBlock 4</td>
 +
    <td>2040</td>
 +
    <td>8.7 </td>
 +
    <td>0.05</td>
 +
    <td>63.03</td>
 +
    <td>7.24</td>
 +
  </tr>
 +
  <tr>
 +
    <td>gBlock 5</td>
 +
    <td>723</td>
 +
    <td>36.5 </td>
 +
    <td>0.05</td>
 +
    <td>22.34</td>
 +
    <td>0.61</td>
 +
  </tr>
 +
  <tr>
 +
    <td>pSB1C3</td>
 +
    <td>2070</td>
 +
    <td>25 [stock] </td>
 +
    <td>0.05</td>
 +
    <td>63.96</td>
 +
    <td>2.56</td>
 +
  </tr>
 +
 
 +
 
 +
</table>
 +
 
 +
<h6> 1. Set up the following reaction on ice:</h6>
 +
<ul>
 +
  <table style="width:80%">
 +
  <tr>
 +
    <th> </th>
 +
    <th>4-6 Fragment Assembly</th>
 +
    <th>Positive Control</th>
 +
  </tr>
 +
  <tr>
 +
    <td>Recommended DNA Ratio</td>
 +
    <td>vector:insert = 1:1</td>
 +
    <td> </td>
 +
  </tr>
 +
  <tr>
 +
    <td>Total Amount of Fragments</td>
 +
    <td> 0.2-0.5 pmols X uL
 +
      <ul>1.84 uL of gBlock 1</ul>
 +
        <ul>5.84 uL of gBlock 2</ul>
 +
      <ul>2.00 uL of gBlock 3</ul>
 +
      <ul>7.24 uL of gBlock 4</ul>
 +
      <ul>0.61 uL of gBlock 5</ul>
 +
      <ul>2.56 uL of pSB1C3</ul>
 +
     
 +
    </td>
 +
    <td>10 uL</td>
 +
 
 +
  </tr>
 +
  <tr>
 +
    <td>NEBuilder HiFi DNA Assembly Master Mix</td>
 +
    <td>20 uL</td>
 +
    <td>10 uL</td>
 +
 
 +
  </tr>
 +
  <tr>
 +
    <td>Total Volume</td>
 +
    <td>40.09 uL</td>
 +
    <td>20 uL </td>
 +
  </tr>
 +
 
 +
 
 +
</table>
 +
 
 +
</ul>
 +
<h6> 2. Incubate samples in a thermocycler at 50 degree Celsius for 60 minutes. Following incubation, store samples on ice or at -20 degree Celsius for subsequent transformation. </h6>
 +
<h6> 3. Thaw DH10B competent cells on ice.</h6>
 +
<h6> 4. Add 2 uL of the chilled assembled product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4-5 times. Do not vortex.</h6>
 +
<h6> 5. Place the mixture on ice for 30 minutes. Do not mix.</h6>
 +
<h6> 6. Heat shock at 42 degree Celsius for 1.5 minutes. Do not mix.</h6>
 +
<h6> 7. Transfer tubes to ice for 4 minutes.</h6>
 +
<h6> 8. Add 1 mL of LB-Ara-Trp to the tube. LB-Ara-Trp can be prepared by adding 200 uL of 20% arabinose stock, 0.23 g of tryptophan, and then LB to a final volume of 20 mL.</h6>
 +
<h6> 9. Incubate the tube at 37 degree Celsius for 60 minutes. Shake vigorously (250 rpm) or rotate.</h6>
 +
<h6> 10. Warm CM30 plate to 37 degree Celsius after spreading the plate with 100 uL of 20% arabinose stock and 100 uL of 0.1% tryptophan stock.</h6>
 +
<h6> 11. Centrifuge the cells and discard 1 mL of supernatant. Use the remaining 100 uL to resuspend the pellet and spread the 100 uL of the cells onto the CM30 plate. Use Amp plate for positive control sample.</h6>
 +
<h6> 12. Incubate overnight at 37 degree Celsius.</h6>
 +
 
 +
</ul>
 +
 
 +
</ul>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<table width="100%"  cellspacing="0" height="200px" border = "0"> </table>
 +
           
 +
 
 +
 
 +
        </div>
 +
      </div>
 +
    </div>
 +
  </body>
 
</html>
 
</html>

Revision as of 22:40, 18 September 2015

RESULT

Design and Order gBlock

After the construction and optimization of our killing switch system that aims to work in E. coli BL21(DE3), we distribute them into 5 gBlock fragments, a synthetic double-stranded DNA product provided by IDT company. This strategy is chosen since our design included several new parts such as Cas9 and guiding RNA that target specific sequences. The whole construct also involved in numerous minor changes, thus traditional cloning and assembly of available biobricks cannot meet our needs.

Fig 1. Span of gBlock corresponding to the killing switch construct. For easy understanding, the length of DNA fragments in the figure are not in actual proportion.

These gBlocks are DNA fragments synthesized according to our order, and can be later assembled by Hi-Fi assembly. Fig. 1 shows the span of the gBlock fragments and their length in base pair. gBlock 1 initiates with prefix and gblock 5 ends with suffix, thus after assembly with pSB1C3 backbone, the whole construct forms a circular plasmid and can be transformed into targeted organisms.

Design and Order gBlock Primer

Fig. 2 Relative position of primers and gBlocks

While ordering the gBlock DNAs, we also designed and ordered the 10 forward and reverse primers that matched the 5 gBlocks respectively in order to amplify them through PCR. Fig. 2 illustrates the relative positions of gBlocks and primers in a circular plasmid. Since the forward/ reverse primers have overlapping regions on the preceding/ following gBlocks, after amplification all fragments would have overlapping sequences that enable Hi-Fi assembly. The sequences and working temperatures of each primers are provided in the table below. All primers were designed to have around 40 base pairs to provide enough specificity for the annealing DNA. Yet the arrangement exceeded the optimal length of primer design (which should be 18-24 bp) and the primers might have a higher chance to generate self-dimers or form secondary structure.

Overlaps Oligo (Uppercase = gene-specific primer) 5'->3' Anneals F/R 3' Tm 3' Ta
gBlock 4 tgggcctttctgcgtttataTACTAGAGTTTACGGCTAG gBlock 5 Fwd 55.1°C 58.1°C
pSBCc3 tgcagcggccgctactagtaAAAAAAAGCACCGACTCG gBlock 5 Rev 58.3°C 58.1°C
pSB1C3 attcgcggccgcttctagagTTATGACAACTTGACGGC gBlock 1 Fwd 57.1°C 58.4°C
gBlock 2 cgtgcaaataatcaatgtctCTAGTAATAGCAAAGTGTGAC gBlock 1 Rev 55.4°C 58.4°C
gBlock 3 aactgtcaaagttgttgatgAATTAGTAAAAGTGATGGGTCG gBlock 4 Fwd 57.7°C 59.9°C
gBlock 5 gctagccgtaaactctagtaTATAAACGCAGAAAGGCC gBlock 4 Rev 56.7°C 59.9°C
gBlock 2 caatctgatcgcattgtcgcTGGGTCTGACCCCTAACT gBlock 3 Fwd 62.5°C 60.9°C
gBlock 4 acccatcacttttactaattCATCAACAACTTTGACAGTTTG gBlock 3 Rev 57.9°C 60.9°C
gBlock 1 tcacactttgctattactagAGACATTGATTATTTGCACGG gBlock 2 Fwd 58.5°C 61.5°C
gBlock 3 aaagttaggggtcagacccaGCGACAATGCGATCAGATTG gBlock 2 Rev 62.3°C 61.5°C
--- CTCTAGAAGCGGCCGCGA pSB1C3 Rev 68.1°C 67.5°C
--- TACTAGTAGCGGCCGCTG pSB1C3 Fwd 64.5°C 67.5°C
Table 1. Sequences of the 10 ordered forward and reverse primers. The lowercase letters correspond to the overlapping sequences and the capital letters represent the annealing sequences of the adjacent gBlock fragments. Tm stands for melting temperature, and Ta stands for annealing temperature.
4. If Taq polymerase is used instead of Q5, assemble all reaction components as followed on ice. Gently mix all the components before use. Collect all liquid to the bottom of the tube by a quick spin if necessary.
    COMPONENT 50 uL REACTION
    Taq buffer 5 uL
    dNTPs 1 uL
    10 uM Forward Primer 1 uL
    10 uM Reverse Primer 1 uL
    Taq polymerase 0.5 uL
    Template gBlock DNA 2 uL
    PCR water 39.5 uL
5. Transfer PCR tubes to a PCR machine and begin thermocycling.
    STEP TEMPERATURE TIME
    Initial Denaturation 98 degree Celsius 30 second
    30 cycles 98 degree Celsius 10 seconds
    50-72 degree Celsius 30 seconds
    72 degree Celsius 1.5 minutes
    Final Extension 72 degree Celsius 2.5 minutes
    Hold 4 degree Celsius

Gel Extraction

    - QIAquick Gel Extraction Kit

      1. Cut the specific DNA fragments from the agarose gel with a scalpel.
      2. Weigh the gel slice in an Eppendorf tube. Add 3 volumes buffer QG to 1 volume gel (100 mg gel to 100 uL).
      3. Incubate the tube at 50 degree Celsius for 10 minutes. Vortex the tube every 2-3 minutes to help dissolve gel. The colour of the mixture is yellow.
      4. Add 1 gel volume isopropanol to the sample and mix.
      5. Place a QIAquick column in s provided 2-mL collection tube. Apply the sample to the QIAquick column and centrifuge for 1 minute. Discard the flow-through and place back the column back into the same tube (binding DNA).
      6. Add 750 uL of buffer PE/EtOH to QIAquick column. Let the column stand for 4 minutes and centrifuge for 1 minute. Discard the flow-through and place back the QIAquick column back into the same tube (washing). Centrifuge the QIAquick column in the provided 2-mL collection tube for 1 minute to remove residual wash buffer.
      7. Place the QIAquick column into a clean Eppendorf tube.
      8. Add 30 uL of PCR water to the centre of the QIAquick membrane to elute DNA. Let the column stand for 1 minute and centrifuge for 1 minute.

HiFi DNA Assembly and Transformation

    - NEBuilder HiFi DNA Assembly Master Mix

    - Positive control provided: 2 overlapping dsDNA fragments for control assembly

      length (bp) Conc. (ng/uL) pmol needed Mass (ng) Vol. (uL)
      gBlock 1 1160 19.5 0.05 35.84 1.84
      gBlock 2 1889 10 [stock] 0.05 58.37 5.84
      gBlock 3 1540 23.8 0.05 47.58 2.00
      gBlock 4 2040 8.7 0.05 63.03 7.24
      gBlock 5 723 36.5 0.05 22.34 0.61
      pSB1C3 2070 25 [stock] 0.05 63.96 2.56
      1. Set up the following reaction on ice:
        4-6 Fragment Assembly Positive Control
        Recommended DNA Ratio vector:insert = 1:1
        Total Amount of Fragments 0.2-0.5 pmols X uL
          1.84 uL of gBlock 1
          5.84 uL of gBlock 2
          2.00 uL of gBlock 3
          7.24 uL of gBlock 4
          0.61 uL of gBlock 5
          2.56 uL of pSB1C3
        		 10 uL
        NEBuilder HiFi DNA Assembly Master Mix 20 uL 10 uL
        Total Volume 40.09 uL 20 uL
      2. Incubate samples in a thermocycler at 50 degree Celsius for 60 minutes. Following incubation, store samples on ice or at -20 degree Celsius for subsequent transformation.
      3. Thaw DH10B competent cells on ice.
      4. Add 2 uL of the chilled assembled product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4-5 times. Do not vortex.
      5. Place the mixture on ice for 30 minutes. Do not mix.
      6. Heat shock at 42 degree Celsius for 1.5 minutes. Do not mix.
      7. Transfer tubes to ice for 4 minutes.
      8. Add 1 mL of LB-Ara-Trp to the tube. LB-Ara-Trp can be prepared by adding 200 uL of 20% arabinose stock, 0.23 g of tryptophan, and then LB to a final volume of 20 mL.
      9. Incubate the tube at 37 degree Celsius for 60 minutes. Shake vigorously (250 rpm) or rotate.
      10. Warm CM30 plate to 37 degree Celsius after spreading the plate with 100 uL of 20% arabinose stock and 100 uL of 0.1% tryptophan stock.
      11. Centrifuge the cells and discard 1 mL of supernatant. Use the remaining 100 uL to resuspend the pellet and spread the 100 uL of the cells onto the CM30 plate. Use Amp plate for positive control sample.
      12. Incubate overnight at 37 degree Celsius.