Difference between revisions of "Team:SCUT China/Notebook"

 
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<div class="mainContent">
 
<div class="mainContent">
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  <div class="experiments"></div>
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  <div class="notebook"></div>
 
   <div class="container-text" style="font-size:18px">
 
   <div class="container-text" style="font-size:18px">
  
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       <p><strong><span style="font-family:'微软雅黑',sans-serif; font-size:18.0pt; ">Our</span></strong>&nbsp;&nbsp;team have seven wet lab experimenters, all of whom are laboratory rookies at first. We still remembered how hard for us to spend about 3 months completing the first plasmid construction. After one year iGEM training, we are skilled in molecular cloning operation - can complete multiple plasmids construction simultaneously in 3 days at the soonest now. During this time, many experiments failures we have experienced. But we have got a lot of joy from the success of the experiments at the same time. Through the training for the whole project, we deeply understand the importance of laboratory safety and strict implementation of protocols when performing an experiment.   
+
       <p style=" font-size:18px;font-weight:500px;line-height:27px;text-align:justify">Our team have seven wet lab experimenters, all of whom are laboratory rookies at first. We still remembered how hard for us to spend about 3 months completing the first plasmid construction. After one year iGEM training, we are skilled in molecular cloning operation - can complete multiple plasmids construction simultaneously in 3 days at the soonest now. During this time, many experiments failures we have experienced. But we have got a lot of joy from the success of the experiments at the same time. Through the training for the whole project, we deeply understand the importance of laboratory safety and strict implementation of protocols when performing an experiment.   
 
Consequently, we would like to post our protocols detailed enough for other team to repeat our experiments.  Simply click on a protocol to read its steps. The side menu to scroll to the specific protocol is convenient for you to look for.
 
Consequently, we would like to post our protocols detailed enough for other team to repeat our experiments.  Simply click on a protocol to read its steps. The side menu to scroll to the specific protocol is convenient for you to look for.
 
</p>
 
</p>
 
   </div>
 
   </div>
 
     <section class="popout protocols">
 
     <section class="popout protocols">
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       <div class="collapsible" id="Agarplates"> Basic experiments</div>
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       <div class="collapsible" id="Agarplates"> Modification of chassis </div>
 
       <div class="collapsibleContent">
 
       <div class="collapsibleContent">
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        <p class="MsoNormal"><span style="font-size:14.0pt; ">(1)Lysogeny Broth  (LB) </span></p>
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                <center> <object class="file-pdf"data="https://static.igem.org/mediawiki/2019/3/3e/T--SCUT_China--Modification_of_chassis.pdf
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Formula: </span></p>
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">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">NaCl 1%</span></p>
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          <embed src="https://static.igem.org/mediawiki/2019/3/3e/T--SCUT_China--Modification_of_chassis.pdf" type="application/pdf">
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Tryptone 1%</span></p>
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        </object></center>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Yeast extract  0.5%</span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Agar 2% (Solid)</span></p>
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              </div>
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<p class="MsoNormal"><span style="font-size:14.0pt; ">(2)Antibiotic-concentrations:</span></p>
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<table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="559" style="border-collapse:collapse;border:none;">
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  <tr style="height:14.7pt;">
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    <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.7pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Antibiotic</span></p></td>
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    <td width="138" valign="top" style="width:103.3pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.7pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Formula </span></p></td>
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−
    <td width="153" valign="top" style="width:115.0pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.7pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Standard Concentration</span></p></td>
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    <td width="153" valign="top" style="width:114.6pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.7pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Working Concentration</span></p></td>
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−
  </tr>
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−
  <tr>
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−
    <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Ampicillin </span></p></td>
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−
    <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">1g+10mL ddH<sub>2</sub>O</span></p></td>
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−
    <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">100mg/mL</span></p></td>
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−
    <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">25-50</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
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−
  </tr>
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−
  <tr>
+
−
    <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">kanamycin</span></p></td>
+
−
    <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">0.1g+10mL ddH<sub>2</sub>O</span></p></td>
+
−
    <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">10mg/mL</span></p></td>
+
−
    <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">10-50</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Chloramphenicol</span></p></td>
+
−
    <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">0.25g+10mL ethanol</span></p></td>
+
−
    <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">100mg/mL</span></p></td>
+
−
    <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">2.5-25</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Carbenicillin</span></p></td>
+
−
    <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">1g+10mL ddH<sub>2</sub>O</span></p></td>
+
−
    <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">100mg/mL</span></p></td>
+
−
    <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">25-50</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Tetracycline </span></p></td>
+
−
    <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">0.1g+10mL ddH<sub>2</sub>O</span></p></td>
+
−
    <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">10mg/mL</span></p></td>
+
−
    <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">10-50</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
+
−
  </tr>
+
−
</table>
+
−
<p class="MsoNormal">&nbsp;</p>
+
−
<p class="MsoNormal"><span style="font-size:14.0pt; ">(3)Filtration  Sterilization</span></p>
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−
<p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Acrodisc 32mm syringe filters with 0.2μm  Supor membrane are used to remove bacteria from liquids.</span></p>
+
−
<p class="MsoNormal"><span style="font-size:14.0pt; ">(4)Culture in  Incubator&amp;in Shaker</span></p>
+
−
<p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">In Incubator: at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ </span></p>
+
−
<p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">In Shaker: at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm</span><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; "> </span></strong></p>
+
−
<p class="MsoNormal"><span style="font-size:14.0pt; ">(5)Glycerol Stock</span></p>
+
−
<p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Add 250</span><span style="font-family:'微软雅黑',sans-serif; ">μl 60% glycerol to 750μl  bacteria solution and mix them thoroughly.</span></p>
+
−
<p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Store at -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; "> </span></strong></p>
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−
      </div>
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     </section>
 
     </section>
  
 
     <section class="popout protocols">
 
     <section class="popout protocols">
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       <div class="collapsible" id="Competence">Plasmid Extraction</div>
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       <div class="collapsible" id="Competence">Verification of acid tolerant factors</div>
 
       <div class="collapsibleContent">
 
       <div class="collapsibleContent">
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Add ethanol (96-100%) to Buffer PW before use, check  bottle tag for the adding volume. </span></p>
+
              <center> <object class="file-pdf"data="https://static.igem.org/mediawiki/2019/a/a1/T--SCUT_China--Verification_of_acid_tolerant_factors.pdf">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Column equilibration: Place a Spin Column CP3 in  a clean collection tube, and add 500 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer BL to CP3. Centrifuge for  1 min at 12,000 rpm (~13,400 × g) in a table-top  microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back  into the collection tube. (Please use freshly treated spin column). </span></p>
+
          <embed src="https://static.igem.org/mediawiki/2019/a/a1/T--SCUT_China--Verification_of_acid_tolerant_factors.pdf" type="application/pdf">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Harvest 1-5 ml bacterial cells in a  microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) in a conventional, table-top microcentrifuge for 1 min at room  temperature (15- 25°C), then remove all traces of  supernatant by inverting the open centrifuge tube until all medium has been  drained (For large volume of bacterial cells, please harvest to one tube by  several centrifugation step.) </span></p>
+
         </object></center>
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(3) Re-suspend the bacterial pellet in 250 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer P1 (Ensure that RNase A has been added). The bacteria  should be resuspended completely by vortex or pipetting up and down until no  cell clumps remain. </span></p>
+
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: No cell clumps should be visible after  resuspension of the pellet, otherwise incomplete lysis will lower yield and  purity. </span></p>
+
            </div>
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(4) Add 250 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer P2 and mix gently and  thoroughly by inverting the tube 6-8 times. Note: Mix gently by inverting the  tube. Do not vortex, as this will result in shearing of genomic DNA. If  necessary, continue inverting the tube until the solution becomes viscous and  slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.  If the lysate is still not clear, please reduce bacterial pellet. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(5) Add 350 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer P3 and mix immediately  and gently by inverting the tube 6-8 times. The solution should become cloudy.  Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Note: To avoid localized precipitation, mix the  solution thoroughly, immediately after addition of Buffer P3. If there is still  white precipitation in the supernatant, please centrifuge again. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(6) Transfer the supernatant from step 5 to the Spin  Column CP3 (place CP3 in a collection tube) by decanting or pipetting.  Centrifuge for 30-60 s at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g). Discard the  flow-through and set the Spin Column CP3 back into the Collection Tube. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(7) (Optional) Wash the Spin Column CP3 by adding  500 µl Buffer PD and centrifuge for 30-60 s at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g). Discard the flow-through and put Spin Column CP3 back to the  collection tube. This step is recommended to remove trace nuclease activity  when using endA<sup>+</sup> strains such as the JM series, HB101 and its  derivatives, or any wild-type strain, which have high levels of nuclease  activity or high carbohydrate content. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(8) Wash the Spin Column CP3 by adding 600 µl Buffer  PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 30-60 s  at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g). Discard the flow-through, and  put the Spin Colum CP3 back into the Collection Tube. </span></p>
+
−
         <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(9) Repeat Step 8. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(10) Centrifuge for an additional 2 min at 12,000  rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) to remove residual wash Buffer PW. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: Residual ethanol from Buffer PW may inhibit  subsequent enzymatic reactions. We suggest open CP3 lid and stay at room  temperature for a while to get rid of residual ethanol. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(11) Place the Spin Column CP3 in a clean 1.5 ml  microcentrifuge tube. To elute DNA, add 50-100 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer EB to  the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min  at 12,000 rpm (~13,400 × g). </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: If the volume of eluted buffer is less than 50 </span><span style="font-family:'微软雅黑',sans-serif; ">μl, it may affect recovery efficiency. The pH value of eluted buffer  will have some influence in eluting; Buffer EB or distilled water (pH 7.0-8.5)  is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in  Buffer EB and storing at -20°C is recommended, since  DNA stored in water is subject to acid hydrolysis. Repeat step 11 to increase plasmid  recovery efficiency.</span></p>
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−
      </div>
+
 
     </section>
 
     </section>
  
 
     <section class="popout protocols">
 
     <section class="popout protocols">
−
       <div class="collapsible"> Bacterial Genome Extraction</div>
+
       <div class="collapsible"> Verification of Toehold Switches</div>
 
       <div class="collapsibleContent">
 
       <div class="collapsibleContent">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Ensure that Buffer GD and Buffer PW have been prepared with appropriate volume of ethanol (96-100%)  as indicated on the bottle and shake thoroughly. </span></p>
+
         
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Pipet 1-5 ml  bacterial culture suspension in a centrifuge tube, centrifuging for 1 min at  10,000 rpm (~11,500 </span><span style="font-family:'微软雅黑',sans-serif; ">× g), discard  supernatant as possible. </span></p>
+
<table align="center">  
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Add 200 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer GA. Mix thoroughly by vortex. </span></p>
+
<object class="file-pdf"data="https://static.igem.org/mediawiki/2019/4/44/T--SCUT_China--Verification.pdf
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: For  difficult-broken Gram-positive bacteria, Step 2 could be replaced by lysozyme  treatment: add 180 </span><span style="font-family:'微软雅黑',sans-serif; ">μl enzymatic  lysis buffer (20 mM Tris·Cl, pH 8.0; 2 mM sodium EDTA;  1.2% Triton<sup>®</sup> X-100; lysozyme (final concentration of 20 mg/ml)).  Incubate for at least 30 min at 37°C. Lysozyme should  be prepared with buffer, otherwise the lysozyme is not active. If RNA-Free  genomic DNA is required, add 4 μl RNase A (100 mg/ml,  should be prepared by user, Cat. no. RT405-12), mix by vortex for 15 s, and  incubate for 5 min at room temperature (15- 25°C). </span></p>
+
">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(3) Add 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Proteinase K. Mix thoroughly by vortex. </span></p>
+
          <embed src="https://static.igem.org/mediawiki/2019/4/44/T--SCUT_China--Verification.pdf
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(4) Add 220 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer GB to the sample, vortex for 15 s, and  incubate at 70°C for 10 min to yield a homogeneous  solution. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from  the inside of the lid. </span></p>
+
" type="application/pdf">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: White  precipitates may form when Buffer GB is added. They will not interfere with the  procedure and will dissolve during the heat incubation at 70</span><span style="font-family:'微软雅黑',sans-serif; ">°C. If precipitates do not dissolve during heat  incubation, it indicates that the cell is not completely lysed and may result  in low yield of DNA and impurity in DNA. </span></p>
+
         </object>
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(5) Add 220 </span><span style="font-family:'微软雅黑',sans-serif; ">μl ethanol (96-100%) to the sample, and mix thoroughly  by vortex for 15 s. A white precipitate may form on addition of ethanol. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from the inside  of the lid. </span></p>
+
        </table>
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(6) Pipet the mixture  from step 5 into the Spin Column CB3 (in a 2 ml collection tube) and centrifuge  at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g)  for 30 s. Discard flow-through and place the spin column into the collection  tube. </span></p>
+
              </div>
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(7) Add 500 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer GD (Ensure ethanol (96-100%) has been added)  to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s, then discard the flow-through and place the spin  column into the collection tube. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(8) Add 600 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer PW (Ensure ethanol (96-100%) has been added)  to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s. Discard the flow-through and place the spin column  into the collection tube. </span></p>
+
−
         <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(9) Repeat Step 8. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(10) Centrifuge at  12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) for 2 min to  dry the membrane completely. Note: The residual ethanol of buffer PW may affect  downstream application.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(11) Place the Spin  Column CB3 in a new clean 1.5 ml centrifuge tube, and pipet 50-200 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer TE or distilled water directly to the center  of the membrane. Incubate at room temperature (15-25°C)  for 2-5 min, and then centrifuge for 2 min at 12,000 rpm (~13,400 × g). </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: If the volume of  eluted buffer is less than 50 </span><span style="font-family:'微软雅黑',sans-serif; ">μl,  it may affect recovery efficiency. What&rsquo;s more, the pH  value of eluted buffer will have some influence in eluting, we suggest chose  buffer TE or distilled water (pH 7.0-8.5) to elute gDNA. For longterm storage  of DNA, eluting in Buffer TE and storing at -20°C is  recommended, since DNA stored in water is subject to acid hydrolysis.</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
+
−
      </div>
+
 
     </section>
 
     </section>
  
 
     <section class="popout protocols">
 
     <section class="popout protocols">
−
       <div class="collapsible" id="GenDNA">Primer Design</div>
+
       <div class="collapsible" id="GenDNA">Improvement of T7 promoters</div>
 
       <div class="collapsibleContent">
 
       <div class="collapsibleContent">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">All Primers used in experiments meet basic requirements below:</span></p>
+
 
−
        <ol>
+
<table align="center">  
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">Free 3</span><span style="font-family:'微软雅黑',sans-serif; ">&rsquo;-end.</span></li>
+
<object class="file-pdf"data="https://static.igem.org/mediawiki/2019/3/36/T--SCUT_China--Improvement_of_T7_promoters.pdf">
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">Number of Mismatches&lt;150.</span></li>
+
          <embed src="https://static.igem.org/mediawiki/2019/3/36/T--SCUT_China--Improvement_of_T7_promoters.pdf" type="application/pdf">
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">55</span><span style="font-family:'微软雅黑',sans-serif; ">℃&lt;T<sub>m</sub>&lt;72℃ </span></li>
+
         </object>
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">18 bp&lt;Length&lt;25 bp</span></li>
+
        </table>
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">40%&lt;GC Content&lt;60%</span></li>
+
              </div>
−
        </ol>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The Softwares Oligo7  and SnapGene are used to design primers.</span></p>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">Especially:</span></strong></p>
+
−
        <ol>
+
−
          <li><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">For Gibson Assembly</span></strong></li>
+
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">68</span><span style="font-family:'微软雅黑',sans-serif; ">℃&lt;Overhand  T<sub>m</sub>&lt;72℃ </span></li>
+
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">45%&lt;GC Content&lt;55%</span></li>
+
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">The difference between T<sub>m</sub> of overhand of each  primer is no more than 2</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span></li>
+
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">The length of part combining with targeted fragment  is between 18bp and 25bp.</span></li>
+
−
          <li><span style="font-family:'微软雅黑',sans-serif; ">The difference between T<sub>m</sub> of combining  parts of two primers is no more than 5</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span></li>
+
−
          <li><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">For sequencing</span></strong></li>
+
−
         </ol>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; The length  is around 20bp.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; The number  of hairpin structures and primer dimers is less than 3 respectively. And the number  of mismatches is strictly less than 100.</span></p>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(3) For overlap PCR</span></strong></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">55</span><span style="font-family:'微软雅黑',sans-serif; ">℃&lt;T<sub>m</sub> of each primer&lt;60℃ </span></p>
+
−
      </div>
+
 
     </section>
 
     </section>
  
 
   <section class="popout protocols">
 
   <section class="popout protocols">
−
       <div class="collapsible" id="plasmidDNA">Polymerase Chain Reaction (PCR)</div>
+
       <div class="collapsible" id="plasmidDNA">Construction of VerProS pool</div>
 
       <div class="collapsibleContent">
 
       <div class="collapsibleContent">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Primers preparation:  Carefully hold the lyophilized primer powder and centrifuge at 12000rmp for  2min. Dilute to 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μM with ddH<sub>2</sub>O.</span></p>
+
      <table align="center">  
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Q5 PCR</span></strong></p>
+
<object class="file-pdf"data="https://static.igem.org/mediawiki/2019/0/0f/T--SCUT_China--Construction_of_VerProS_pool.pdf">
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="581" style="border-collapse:collapse;border:none;">
+
           <embed src="https://static.igem.org/mediawiki/2019/0/0f/T--SCUT_China--Construction_of_VerProS_pool.pdf" type="application/pdf">
−
          <tr>
+
         </object>
−
            <td width="241" colspan="2" valign="top" style="width:180.8pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><a name="_Hlk16355836"><span style="font-family:'微软雅黑',sans-serif; ">10</span></a><span style="font-family:'微软雅黑',sans-serif; ">μl    Assay</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
        </table>
−
            <td width="128" valign="top" style="width:96.1pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
+
−
            <td width="117" valign="top" style="width:87.95pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span><span style="font-family:'微软雅黑',sans-serif; ">×T5 Reaction Buffer</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
+
−
            <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial Denaturation</span></p></td>
+
−
            <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30 s</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">dNTPs </span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.8</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
+
−
            <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10 s</span></p></td>
+
−
            <td width="47" rowspan="3" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer </span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.25</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
+
−
            <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30 s</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Reverse primer</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.25</span></p></td>
+
−
            <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
+
−
            <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Dependent</span></p></td>
+
−
            <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final Elongation</span></p></td>
+
−
            <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2 min</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Q5 polymerase</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.1</span></p></td>
+
−
            <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
+
−
            <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
+
−
            <td width="47" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Dependent</span></p></td>
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            <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
          </tr>
+
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        </table>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; The temperature of annealing=T<sub>m</sub> of primers-5</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span>        </p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; The final concentration of each template is 1ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μl.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; The time of elongation depends on the length of fragments to be  amplified:</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1 kb: 15 s-30 s</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">≥1 kb: 40 s/kb</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">≥2.5 kb: 1 min/kb</span></p>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) T5 PCR</span></strong></p>
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="568" style="border-collapse:collapse;border:none;">
+
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          <tr>
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−
            <td width="241" colspan="2" valign="top" style="width:180.8pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">μl Assay</span></p></td>
+
−
            <td width="121" valign="top" style="width:90.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
+
−
            <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.65pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
+
−
            <td width="44" valign="top" style="width:33.3pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">×T5 Super PCR Mix (Colony)</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span></p></td>
+
−
            <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial denaturation</span></p></td>
+
−
            <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
+
−
            <td width="44" valign="top" style="width:33.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer </span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span></p></td>
+
−
            <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
+
−
            <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10s</span></p></td>
+
−
            <td width="44" rowspan="3" valign="top" style="width:33.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">29</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Feverse primer </span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span></p></td>
+
−
            <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
+
−
            <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T<sub>m</sub>-5</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
+
−
           </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
+
−
            <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
+
−
            <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s/kb</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2.6</span></p></td>
+
−
            <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final elongation</span></p></td>
+
−
            <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2min</span></p></td>
+
−
            <td width="44" valign="top" style="width:33.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
+
−
            <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
+
−
            <td width="44" valign="top" style="width:33.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
        </table>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(3) Overlap PCR</span></strong></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step 1:</span></p>
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="538" style="border-collapse:collapse;border:none;">
+
−
          <tr>
+
−
            <td width="179" colspan="2" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">25</span><span style="font-family:'微软雅黑',sans-serif; ">μl Assay</span></p></td>
+
−
            <td width="132" valign="top" style="width:99.2pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.15pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.5pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Buffer</span></p></td>
+
−
            <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span></p></td>
+
−
            <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial Denaturation</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">dNTPs</span></p></td>
+
−
            <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
+
−
            <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10s</span></p></td>
+
−
            <td width="47" rowspan="3" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template 1</span></p></td>
+
−
            <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Dependent</span></p></td>
+
−
            <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T<sub>m</sub>-5</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template 2</span></p></td>
+
−
            <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Dependent</span></p></td>
+
−
            <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s/kb</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Enzyme</span></p></td>
+
−
            <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.25</span></p></td>
+
−
            <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final Elongation</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2min</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
+
−
            <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
         </table>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">*The final  concentration of each template is 50ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μl.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step 2:</span></p>
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="557" style="border-collapse:collapse;border:none;">
+
−
          <tr>
+
−
            <td width="165" colspan="2" valign="top" style="width:123.55pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">50</span><span style="font-family:'微软雅黑',sans-serif; ">μl Assay (μl)</span></p></td>
+
−
            <td width="166" valign="top" style="width:124.3pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.1pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.55pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Buffer</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span></p></td>
+
−
            <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial Denaturation</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">dNTPs</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
+
−
            <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10s</span></p></td>
+
−
            <td width="47" rowspan="3" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">25</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1.25</span></p></td>
+
−
            <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T<sub>m</sub>-5</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Reverse primer</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1.25</span></p></td>
+
−
            <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s/kb</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">25</span></p></td>
+
−
            <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final Elongation</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2min</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Enzyme</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.25</span></p></td>
+
−
            <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH2O</span></p></td>
+
−
            <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">15.25</span></p></td>
+
−
            <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
          </tr>
+
−
        </table>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(4) Colony PCR</span></strong></p>
+
−
<p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Pick several  colonies and transfer them respectively to different grids (drew outside the  bottom of the dish) of a new culture plate.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Culture the  bacteria for 8-10h.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Add 20</span><span style="font-family:'微软雅黑',sans-serif; ">μl ddH<sub>2</sub>O to PCR tubes.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Scrape  appropriate amounts of bacteria cells and solve them in ddH<sub>2</sub>O.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Lyse the cells  at 95</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 10min.</span></p>
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="525" style="border-collapse:collapse;border:none;">
+
−
          <tr>
+
−
            <td width="200" colspan="2" valign="top" style="width:150.1pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">μl Assay</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="94" valign="top" style="width:70.25pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
+
−
            <td width="119" valign="top" style="width:89.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
+
−
            <td width="63" valign="top" style="width:47.05pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
+
−
            <td width="49" valign="top" style="width:37.05pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">×T5 Super PCR Mix (Colony)</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial denaturation</span></p></td>
+
−
            <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
+
−
            <td width="49" valign="top" style="width:37.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer </span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
+
−
            <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
+
−
            <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10s</span></p></td>
+
−
            <td width="49" rowspan="3" valign="top" style="width:37.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">29</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Feverse primer </span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
+
−
            <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T<sub>m</sub>-5</span></p></td>
+
−
            <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
+
−
            <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s/kb</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2.6</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
            <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final Elongation</span></p></td>
+
−
            <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
+
−
            <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2min</span></p></td>
+
−
            <td width="49" valign="top" style="width:37.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
+
−
            <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
+
−
            <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
+
−
            <td width="49" valign="top" style="width:37.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
        </table>
+
−
    </div></section>
+
  
−
  <section class="popout protocols">
 
−
      <div class="collapsible">DNA Agarose Gel Electrophoresis    </div>
 
−
    <div class="collapsibleContent">
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Preparation of 1%  gel</span></p>
 
−
      <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="396" style="border-collapse:collapse;border:none;">
 
−
        <tr>
 
−
          <td width="66" valign="top" style="width:49.4pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Size</span></p></td>
 
−
          <td width="85" valign="top" style="width:63.75pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Agarose (g)</span></p></td>
 
−
          <td width="123" valign="top" style="width:92.15pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">×TAE buffer (ml)</span></p></td>
 
−
          <td width="123" valign="top" style="width:92.0pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">DNA stain (</span><span style="font-family:'微软雅黑',sans-serif; ">μl)</span></p></td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td width="66" valign="top" style="width:49.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Big</span></p></td>
 
−
          <td width="85" valign="top" style="width:63.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.8</span></p></td>
 
−
          <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">80</span></p></td>
 
−
          <td width="123" valign="top" style="width:92.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td width="66" valign="top" style="width:49.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Medium</span></p></td>
 
−
          <td width="85" valign="top" style="width:63.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.4</span></p></td>
 
−
          <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">40</span></p></td>
 
−
          <td width="123" valign="top" style="width:92.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td>
 
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        </tr>
 
−
        <tr>
 
−
          <td width="66" valign="top" style="width:49.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Small</span></p></td>
 
−
          <td width="85" valign="top" style="width:63.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span></p></td>
 
−
          <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">20</span></p></td>
 
−
          <td width="123" valign="top" style="width:92.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.5</span></p></td>
 
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        </tr>
 
−
      </table>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Measure out  appropriate amounts of agarose and add corresponding TAE buffer.</span></p>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Heat the  solution until it becomes clear. Add appropriate amounts of DNA stain after it  cools down to about 55</span><span style="font-family:'微软雅黑',sans-serif; ">℃. </span></p>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Pour the  solution to the mold with a comb.</span></p>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Let it  solidify for at least 20min.</span></p>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Move the comb. Put  the gel with the mold in the electrophoresis chamber with TAE buffer.</span></p>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(3) Add 1/10 10</span><span style="font-family:'微软雅黑',sans-serif; ">×Loading buffer to each DNA sample.</span></p>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(4) Pipet samples and  DNA marker to wells.</span></p>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(5) Run for 20-30min  at 120V.</span></p>
 
 
     </div>
 
     </div>
−
  </section>
+
</section>
−
 
+
−
    <section class="popout protocols">
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      <div class="collapsible"> DNA Fragments Recovery</div>
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      <div class="collapsibleContent">
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        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Gel Extraction</span></strong></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Place the  gel under the ultraviolet lamp and quickly cut off the gel containing the  targeted DNA fragments. Remove excess gel as much as possible.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Weigh the  gel and put it in a 1.5 mL centrifuge tube. Add 100</span><span style="font-family:'微软雅黑',sans-serif; ">μL Buffer GDP every 100mg gel.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Water bathe the gels  at 50-55</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 7-10 minutes to dissolve them  completely. During the water bath, turn upside down the tube twice to fasten  dissolution. If the length of DNA fragment is no more than 100bp, add 3 times  volume of Buffer GDP, and equal volume of isopropanol after the water bath.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Centrifuge  briefly to collect droplets on the tube wall. Place a HiPure DNA Column in a 2mL  collection tube. Transfer the solution (</span><span style="font-family:'微软雅黑',sans-serif; ">≤700μL) to the column. Centrifuge for 30-60  seconds at 12,000 × g. If the volume of solution exceeds  700μL, discard the filtrate and put the column back  into the collection tube. Transfer the remaining gels to the column and centrifuge  at 12000 × g for 30-60 seconds.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Discard the  filtrate and put the column back into the collection tube. Add 300 </span><span style="font-family:'微软雅黑',sans-serif; ">μL Buffer GDP to the column. Set it still for a minute.  Centrifuge for 30-60 seconds at 12,000 × g.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Discard the  filtrate and put the column back into the collection tube. Add 600 </span><span style="font-family:'微软雅黑',sans-serif; ">μL Buffer DW2 (has been diluted with absolute ethyl  alcohol) to the column. Centrifuge for 30-60 seconds at 12,000 × g.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;6&gt; Repeat step  &lt;5&gt;.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;7&gt; Discard the  filtrate and put the column back into the collection tube. Centrifuge at 12,000  x g for 2 min. Open the lid of the column and dry for 10-15 minutes in the air  to remove ethanol thoroughly.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;10&gt; Place the  column into a clean 1.5mL centrifuge tube and add 7-30</span><span style="font-family:'微软雅黑',sans-serif; ">μL water (preheated to 55℃) to  the center of the column membrane. Set it still for 2 minutes. Centrifuge at 12,000  × g for 1 min. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;11&gt; Repeat step  &lt;10&gt; to increase yield. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;12&gt; Remove the  column and store the DNA at -20</span><span style="font-family:'微软雅黑',sans-serif; ">℃. </span></p>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) Column Purification</span></strong><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Add ethanol (96-100%)  to Buffer PW before use (see bottle label for volume). All centrifuge steps are  in a conventional tabletop microcentrifuge at room temperature (15-25</span><span style="font-family:'微软雅黑',sans-serif; ">°C).</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Column  equilibration: add 500 Buffer BL to the Spin Column CB1 (put Spin Column CB1  into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) in a table-top microcentrifuge. Discard the  flow-through, and then place Spin Column CB1 back into the collection tube  (please use freshly treated spin column).</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Add 5 volumes  of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix. It  is not necessary to remove mineral oil or kerosene.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: For example, add  250 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer PB to 50 μl PCR reaction (not including oil).</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Transfer the  mixture to the Spin Column CB1, incubate at room temperature (15-25</span><span style="font-family:'微软雅黑',sans-serif; ">°C) for 2 min. Centrifuge for 30-60 s at 12,000 rpm  (~13,400 × g) in a table-top microcentrifuge. Discard  the flow-through, and then place Spin Column CB1 back into the same collection  tube.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: The maximum  loading volume of the column is 800 </span><span style="font-family:'微软雅黑',sans-serif; ">μl. For sample volumes greater than 800 μl  simply load again.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Add 600 µl  Buffer PW (ensure that ethanol (96-100%) has been added) to the Spin Column CB1  and centrifuge for 30-60 s at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g). Discard the flow-through, and place Spin Column  CB1 back in the same collection tube.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: If the purified  DNA is used for the subsequent salt sensitive experiments, such as ligation or  sequencing experiment, it is suggested to stand for 2-5 min after adding Buffer  PW, and then centrifuge.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Repeat step &lt;4&gt;.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;6&gt; Centrifuge  at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g)  for 2 min to remove residual Buffer PW. Discard the flow-through, and allow the  column to air dry with the cap open for several minutes to dry</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">the membrane.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: Residual ethanol  from Buffer PW may inhibit subsequent experiment (enzymatic or PCR reactions).</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;7&gt; Place the  Spin Column CB1 in a clean 1.5 ml microcentrifuge tube. Add 20-50 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer EB to the center of membrane, incubate for 2  min, and centrifuge for 2 min at 12,000 rpm (~13,400 ×  g).</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: If the volume of  eluted buffer is less than 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μl,  it may affect recovery efficiency. The pH value of eluted buffer will have big  influence in eluting; distilled water (pH 7.0-8.5, adjusted with</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">NaOH) is suggested to  elute plasmid DNA, pH&lt;7.0 will decrease elution efficiency. For long-term  storage of DNA, eluting in Buffer EB and storing at -20</span><span style="font-family:'微软雅黑',sans-serif; ">°C is recommended, since DNA stored in</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">water is subject to  acid hydrolysis. Repeat step 7 to increase plasmid recovery efficiency.</span></p>
+
−
      </div>
+
−
  </section>
+
−
 
+
−
    <section class="popout protocols">
+
−
      <div class="collapsible">Gibson Assembly</div>
+
−
      <div class="collapsibleContent">
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Prepare the assay  according to the chart.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Run at 50</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 1h.</span><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; "> </span></strong></p>
+
−
      </div>
+
−
    </section>
+
−
 
+
−
    <section class="popout protocols">
+
−
      <div class="collapsible">Restriction Enzyme Digest</div>
+
−
      <div class="collapsibleContent">
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="568" style="border-collapse:collapse;border:none;">
+
−
          <tr>
+
−
            <td width="307" colspan="2" valign="top" style="width:230.55pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay for single digestion</span></p></td>
+
−
            <td width="261" colspan="2" valign="top" style="width:195.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">50</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay for double digestion</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="176" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">NEB CutSmart<sup>®</sup> Buffer</span></p></td>
+
−
            <td width="131" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
            <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">NEB CutSmart<sup>®</sup> Buffer</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
          </tr>
+
−
          <tr style="height:7.55pt;">
+
−
            <td width="176" rowspan="2" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Restriction Enzyme</span></p></td>
+
−
            <td width="131" rowspan="2" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.5</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
            <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Restriction Enzyme 1</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
          </tr>
+
−
          <tr style="height:7.55pt;">
+
−
            <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Restriction Enzyme 2</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="176" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span></p></td>
+
−
            <td width="131" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Depends on original concentration</span></p></td>
+
−
            <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal" style="margin-left:10.5pt;text-indent:-10.5pt;"><span style="font-family:'微软雅黑',sans-serif; ">Depends on original concentration</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="176" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
+
−
            <td width="131" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="176" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final concentration    of template</span></p></td>
+
−
            <td width="131" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">500ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
            <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final concentration    of template</span></p></td>
+
−
            <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">3000ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="568" colspan="4" valign="top" style="width:425.9pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Incubate at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for several hours.</span></p></td>
+
−
          </tr>
+
−
        </table>
+
−
      </div>
+
−
    </section>
+
−
 
+
−
    <section class="popout protocols">
+
−
      <div class="collapsible">Annealing stitching</div>
+
−
      <div class="collapsibleContent">
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Annealing</span></strong></p>
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="342" style="border-collapse:collapse;border:none;">
+
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          <tr>
+
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            <td width="158" colspan="2" valign="top" style="width:118.4pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">20</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay</span></p></td>
+
−
            <td width="184" colspan="3" valign="top" style="width:138.25pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Procedure</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">×PNK buffer</span></p></td>
+
−
            <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
            <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature</span></p></td>
+
−
            <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
+
−
            <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer</span></p></td>
+
−
            <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
            <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">95</span><span style="font-family:'微软雅黑',sans-serif; ">℃ </span></p></td>
+
−
            <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">3min</span></p></td>
+
−
            <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Reverse primer</span></p></td>
+
−
            <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
            <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">94</span><span style="font-family:'微软雅黑',sans-serif; ">℃ </span></p></td>
+
−
            <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1min</span></p></td>
+
−
            <td width="46" rowspan="2" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">57</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
+
−
            <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">16</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
+
−
            <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">-1</span><span style="font-family:'微软雅黑',sans-serif; ">℃/Cycle</span></p></td>
+
−
            <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
            <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">12</span><span style="font-family:'微软雅黑',sans-serif; ">℃ </span></p></td>
+
−
            <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
+
−
            <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
+
−
          </tr>
+
−
        </table>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) Phosphorylation</span></strong></p>
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="179" style="border-collapse:collapse;border:none;">
+
−
          <tr>
+
−
            <td width="179" colspan="2" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">20</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing product</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">×PNK buffer</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10mM ATP</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T4 PNK enzyme</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
+
−
            <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">14</span></p></td>
+
−
          </tr>
+
−
        </table>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Incubate at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 45min.</span></p>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(3) T4 Ligation</span></strong></p>
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="387" style="border-collapse:collapse;border:none;">
+
−
          <tr>
+
−
            <td width="387" colspan="2" valign="top" style="width:290.35pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Vector plasmid</span></p></td>
+
−
            <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">depends on    concentration</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing product of    promotor</span></p></td>
+
−
            <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing product of    trigger</span></p></td>
+
−
            <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing product of    terminator</span></p></td>
+
−
            <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T4 ligase</span></p></td>
+
−
            <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">×T4 PNK buffer</span></p></td>
+
−
            <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
+
−
            <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></p></td>
+
−
          </tr>
+
−
        </table>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">*The final  concentration of vector in the assay is 100ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μL.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Incubate at 16</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 12h.</span></p>
+
−
      </div>
+
−
    </section>
+
  
 
   <section class="popout protocols">
 
   <section class="popout protocols">
−
    <div class="collapsible">Golden Gate </div>
+
      <div class="collapsible">Construction of working part    </div>
 
     <div class="collapsibleContent">
 
     <div class="collapsibleContent">
−
      <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="549" style="border-collapse:collapse;border:none;">
+
<table align="center">
−
        <tr>
+
<object class="file-pdf"data="https://static.igem.org/mediawiki/2019/2/26/T--SCUT_China--Construction_of_working_part.pdf
−
          <td width="359" colspan="2" valign="top" style="width:269.1pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">20</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay</span></p></td>
+
">
−
          <td width="190" colspan="3" valign="top" style="width:142.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Procedure</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
           <embed src="https://static.igem.org/mediawiki/2019/2/26/T--SCUT_China--Construction_of_working_part.pdf
−
        </tr>
+
" type="application/pdf">
−
        <tr>
+
         </object>
−
          <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid ccdB</span></p></td>
+
        </table>
−
          <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">depends on    concentration</span></p></td>
+
        
−
          <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
           <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
          <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid with Trigger    A mix</span></p></td>
+
−
          <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(As above)</span></p></td>
+
−
          <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">37</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
          <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10min</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
          <td width="46" rowspan="2" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">50</span><span style="font-family:'微软雅黑',sans-serif; ">×</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid with Trigger    B mix</span></p></td>
+
−
          <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(As above)</span></p></td>
+
−
          <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">16</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
          <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">7min</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
         </tr>
+
−
        <tr>
+
−
          <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid with Trigger    C mix</span></p></td>
+
−
          <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(As above)</span></p></td>
+
−
          <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">50</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
          <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10min</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
          <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">×</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid with Trigger    D mix</span></p></td>
+
−
          <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(As above)</span></p></td>
+
−
          <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">12</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
          <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
          <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">×</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Bsal enzyme</span></p></td>
+
−
          <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td>
+
−
          <td style="border:none;padding:0cm 0cm 0cm 0cm;" width="190" colspan="3"><p class="MsoNormal">&nbsp;</td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T4 DNA ligase </span></p></td>
+
−
          <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td>
+
−
          <td style="border:none;padding:0cm 0cm 0cm 0cm;" width="190" colspan="3"><p class="MsoNormal">&nbsp;</td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T4 DNA ligase buffer</span></p></td>
+
−
          <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
+
−
          <td style="border:none;padding:0cm 0cm 0cm 0cm;" width="190" colspan="3"><p class="MsoNormal">&nbsp;</td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH2O</span></p></td>
+
−
          <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; ">&nbsp;</span></strong></p></td>
+
−
          <td style="border:none;padding:0cm 0cm 0cm 0cm;" width="190" colspan="3"><p class="MsoNormal">&nbsp;</td>
+
−
        </tr>
+
−
      </table>
+
−
       <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">*The final  concentration of each plasmid in the assay is 100ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μL.</span></p>
+
 
     </div>
 
     </div>
 
   </section>
 
   </section>
  
 
     <section class="popout protocols">
 
     <section class="popout protocols">
−
       <div class="collapsible" id="GelElectro">Preparation of Competent Cells</div>
+
       <div class="collapsible"> Test of VerProS system</div>
 
       <div class="collapsibleContent">
 
       <div class="collapsibleContent">
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) For Chemical Transformation</span></strong><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
+
<table align="center">  
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Inoculate the bacteria by streaking and culture for 12h.</span></p>
+
<object class="file-pdf"data="https://static.igem.org/mediawiki/2019/5/59/T--SCUT_China--Test_of_VerProS_system.pdf
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Pick a  single colony and dissolve it in in 2mL LB medium. Culture overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm. </span></p>
+
">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Precool the  centrifuge to 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Transfer appropriate amounts of  bacteria solution to fresh medium in the proportion of 1:100. Culture at 220rpm  for some time until OD<sub>600</sub> is between 0.4 and 0.6.</span></p>
+
          <embed src="https://static.igem.org/mediawiki/2019/5/59/T--SCUT_China--Test_of_VerProS_system.pdf
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Transfer the  bacterial cells to cold centrifuge tubes and ice them for 10 minutes to reach 0</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Rotate the tubes sometimes to ensure complete  cooling. </span></p>
+
" type="application/pdf">
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Centrifuge  at 4000 rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 15min.  Discard the supernatant and centrifuge again at 4000rpm and 4℃ for 2 minutes to thoroughly remove the medium.</span></p>
+
         </object>
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;6&gt; Resuspend  cells with 30mL cold and sterilized MgCl<sub>2</sub>-CaCl<sub>2</sub> solution  mildly.</span></p>
+
        </table>
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;7&gt; Centrifuge  at 4000rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> for 15min. Discard the  supernatant and centrifuge again at 4000rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 2 minutes.</span></p>
+
     
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;8&gt; Resuspend  cells with 800</span><span style="font-family:'微软雅黑',sans-serif; ">μL 0.1M cold CaCl<sub>2</sub> mildly. Add 200μL 0.2M 60% glycerol and mix them  thoroughly. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;9&gt; Distribute  the bacteria solution to tubes every 100</span><span style="font-family:'微软雅黑',sans-serif; ">μL.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;10&gt; Froze in  liquid nitrogen for 1min.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;11&gt; Store at  -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span></p>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) For Electrotransformation</span></strong></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Inoculate  the bacteria by streaking and culture for 12h.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Pick a  single colony and dissolve it in in 10mL LB medium. Culture for 8-12h at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Precool the  centrifuge to 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Transfer appropriate amounts of  bacteria culture to fresh medium in the proportion of 1:100. Culture at 37℃ and 220rpm until OD<sub>600</sub> is between 0.4 and 0.6. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Transfer the  bacterial cells to cold centrifuge tubes and ice them for 20-30min. Rotate the  tubes sometimes to ensure complete cooling. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Centrifuge  at 4000 rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 15min.  Discard the supernatant and centrifuge again at 4000rpm and 4℃ for 2min to thoroughly remove the medium. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;6&gt; Suspend and  wash bacterial cells with 25mL cold ultrapure water every two tubes. Centrifuge  at 4000rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 20min. Discard the  supernatant. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;7&gt; Resuspend  cells with 25mL cold 10% glycerol. Centrifuge at 4000 and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃for 20min. Discard the supernatant.</span></p>
+
−
         <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;8&gt; Repeat Step  &lt;7&gt;.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;9&gt; Resuspend  cells with 1mL cold 10% glycerol. Dilute the solution by 1:200. Measure the  concentration and distribute to small tubes every 100</span><span style="font-family:'微软雅黑',sans-serif; ">μL when OD<sub>600</sub> is between 0.5 and 0.7.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;10&gt; Froze in  liquid nitrogen for 1min.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;11&gt; Store at  -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span></p>
+
 
       </div>
 
       </div>
−
    </section>
 
−
 
−
  <section class="popout protocols">
 
−
      <div class="collapsible" id="Transformation">Transformation</div>
 
−
      <div class="collapsibleContent">
 
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Chemical Method</span></strong></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Take  competent cells out of -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃  refrigerator and set on ice for 5-10min to melt them.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Add plasmids  to competent cells solution and flick the tube slightly to mix them up. </span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Set still on  ice for 30min without any vibration.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Heat shock  at 42</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 90s.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Set back on  ice immediately and sit for 2-3min.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;6&gt; Add 800</span><span style="font-family:'微软雅黑',sans-serif; ">μl SOC medium (preheated to 42℃).</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;7&gt; Culture at  37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm for 45-60min.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;8&gt; Centrifuge  at 7000rpm for 3min. Leave 100</span><span style="font-family:'微软雅黑',sans-serif; ">μl </span><span style="font-family:'微软雅黑',sans-serif; ">supernatant  and resuspend the cells.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;9&gt; Spread the bacteria solution on a plate  with antibiotic.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;10&gt; Culture at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 12h.</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
 
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) Electroporation</span></strong></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Take  competent cells out of -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃  refrigerator and unfreeze them for 5-10min on ice.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Add 100-200  ng plasmids to 100</span><span style="font-family:'微软雅黑',sans-serif; ">μL competent  cells solution and flick the tube slightly to mix them up. Set still on ice for  1 min. </span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Suck out all  solution and transfer to a prechilled cuvette. Set still on ice for 5 min.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Set the  cuvette size (2 mm) and voltage (2.5 kV) on the instrument. Insert the cuvette  and deliver the pulse.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Add 1 mL SOC  medium to the cuvette and mix evenly. Transfer all solution to a centrifuge  tube.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;6&gt; Culture at  37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm for 45-60min.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;7&gt; Centrifuge  at 7000rpm for 3min. Leave 100</span><span style="font-family:'微软雅黑',sans-serif; ">μl </span><span style="font-family:'微软雅黑',sans-serif; ">supernatant  and resuspend the cells.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;8&gt; Spread the bacteria solution on a plate  with antibiotic.</span></p>
 
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;9&gt; Culture at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 12h.</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
 
−
      </div></section>
 
−
 
−
  <section class="popout protocols">
 
−
    <div class="collapsible"> Plasmids Discard</div>
 
−
    <div class="collapsibleContent">
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Pick single  colonies from the plate and inoculate in LB liquid medium without antibiotics.  Culture at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm for 12h.</span></p>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Dilute the  bacteria solution by 10<sup>4</sup>, 10<sup>5 </sup>and 10<sup>6</sup> times  successively. For each gradient, spread the solution on two plates with and  without antibiotic respectively.</span></p>
 
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(3) </span><span style="font-family:'微软雅黑',sans-serif; ">Culture  at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 12h.</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
 
−
    </div>
 
 
   </section>
 
   </section>
  
−
  <section class="popout protocols">
+
   </div>
−
    <div class="collapsible"> Red Fluorescence Detection</div>
+
−
    <div class="collapsibleContent">
+
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">TECAN<sup>®</sup> infinite M200 PRO microplate spectrophotometer is used to measure red  fluorescence intensity. The stimulated UV wavelength is 580nm.<strong></strong></span></p>
+
−
    </div>
+
−
  </section>
+
−
 
+
−
  <section class="popout protocols">
+
−
    <div class="collapsible">Optical Density (OD) Measurement</div>
+
−
    <div class="collapsibleContent">
+
−
      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">YOKE<sup>®</sup> UV755B ultraviolet-visible pectrophotometer is used to measure OD under 600nm  tungsten light.</span></p>
+
−
    </div>
+
−
  </section>
+
−
 
+
−
  <section class="popout protocols">
+
−
    <div class="collapsible"> Growth Curve Measurement <i>Streptomyces</i></div>
+
−
  </section>
+
−
 
+
−
  <section class="popout protocols">
+
−
      <div class="collapsible">Screening of the Most Acid-Resistant Strain</div>
+
−
      <div class="collapsibleContent">
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Test Under Acid Pressure</span></strong></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Pick  monoclones from LB plate and inoculate in 2 mL LBG liquid medium (pH 7.0)  containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; After growth  entering stationary phase, measure OD<sub>600</sub>. Dilute bacteria solution  with fresh LBG liquid medium (pH 4.5) until OD<sub>600</sub> value is 1. Transfer 15</span><span style="font-family:'微软雅黑',sans-serif; ">μL diluted liquid to 285 μL LBG  liquid medium (pH 4.5) containing antibiotic on 100-well Honeycomb 2 culture  plate.</span><span style="font-family:'微软雅黑',sans-serif; "> Culture in the  automatic growth curve analyzer Bioscreen C at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and a medium oscillation frequency and amplitude for 24h. Measure  real-time OD<sub>600</sub> every 30min. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Dilute  bacteria solution with certain amounts of LBG fresh liquid medium (pH4.5)  according to the formula: 100 </span><span style="font-family:'微软雅黑',sans-serif; ">× OD<sub>600</sub> value = (100+V) × 1.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Conduct 3-5  parallel experiments.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Procedure:</span></p>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) Test Under Acid Shock</span></strong></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Pick  monoclones from LB plate and inoculate in 2 mL LBG liquid medium (pH 7.0)  containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Transfer the  solution to 10 mL LBG liquid medium (pH 4.5) containing  antibiotic. Incubate at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and  220rpm until OD<sub>600 </sub>value is between 0.5 and 0.6.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Place the  bacteria suspension on ice to stop the growth. Use fresh LBG liquid medium (pH  4.5) to dilute it until OD<sub>600</sub> value is 0.5. Transfer 20</span><span style="font-family:'微软雅黑',sans-serif; ">μL diluted liquid to 180 μL LBG  liquid medium (pH 2.5) containing antibiotic on 96-well plate. Culture at 37℃ and 700rpm in microplate thermostatic oscillator or at 37℃ and 250rpm for 1 h in shaker , and do the control experiment at the  same time. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Dilute the  bacteria solution with fresh LBG liquid medium (pH 4.5) by 10, 10<sup>2</sup>,  10<sup>3</sup> and 10<sup>4</sup> times.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Spread 4 </span><span style="font-family:'微软雅黑',sans-serif; ">μL diluted and undiluted solution on plates  respectively. Culture at room temperature overnight.</span></p>
+
−
        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(3) Glutamate Decarboxylase (GAD)  Activity Test</span></strong></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Pick  monoclones from LB plate and inoculate in 2 mL LB-2% (w/v) glucose liquid  medium containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Transfer the  solution to 10 mL LBG liquid medium (pH 4.5) containing antibiotic. Incubate at  37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm until OD<sub>600 </sub>value  is between 0.5 and 0.6 or incubate for about 16h directly.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Place the  bacteria suspension on ice for 10min to stop the growth. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 3500rpm for 10mim. Discard the supernatant.  Centrifuge again for 2min and remove the medium completely with a pipette.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Suspend  cells with 1mL cold 20 mM K<sub>2</sub>HPO<sub>4</sub>-KH<sub>2</sub>PO<sub>4</sub> (pH7.4) buffer. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and  12000rpm for 2min. Suck out supernatant with a pipette. Centrifuge again, and  remove the supernatant completely with a pipette.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Suspend the  cells with 1mL cold 20 mM K<sub>2</sub>HPO<sub>4</sub>-KH<sub>2</sub>PO<sub>4</sub> (pH7.4) buffer. </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;6&gt; Break up the  cells with a ultrasonic cell grinder. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 12000rpm for 2 min. Take the supernatant crude  enzyme solution for enzyme activity measurement.</span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;7&gt; Measure the  standard growth curves: Mix 10 </span><span style="font-family:'微软雅黑',sans-serif; ">μL  NaOH standard solution preheated to 37℃ and 90 μL of L GAD reaction solution (or blank solution) fully in 96-well  plate. Measure the absorbance with a microplate reader at 37℃. The indicator and corresponding wavelengths are： </span></p>
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="192" style="border-collapse:collapse;border:none;">
+
−
          <tr>
+
−
            <td width="129" valign="top" style="width:96.85pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Bromocresol green</span></p></td>
+
−
            <td width="63" valign="top" style="width:47.5pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">620 nm</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="129" valign="top" style="width:96.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Bromcresol purple</span></p></td>
+
−
            <td width="63" valign="top" style="width:47.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">590 nm</span></p></td>
+
−
          </tr>
+
−
          <tr>
+
−
            <td width="129" valign="top" style="width:96.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Bromothymol blue </span></p></td>
+
−
            <td width="63" valign="top" style="width:47.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">615 nm</span></p></td>
+
−
          </tr>
+
−
        </table>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;8&gt; Measurement  of enzyme activity: Mix 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μL  crude enzyme solution preheated to 37℃ and 180 μL of GAD reaction solution (or blank solution) fully in 96-well  plate. Measure the abosorbance with microplate reader at 37℃ in dynamic detection mode every 1 min for 2 h in the linear range.  The wavelengths are the same as above. Calculate enzyme activity with the  formula below: </span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Enzyme activity  (nmol/min/OD<sub>600</sub>) =<span style="position:relative; top:14.0pt; "><img src="notebook_clip_image002.gif" alt="" width="164" height="46"></span></span></p>
+
−
        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">When:</span></p>
+
−
        <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="568" style="border-collapse:collapse;border:none;">
+
−
          <tr>
+
−
            <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">OD<sub>600</sub></span></p></td>
+
−
            <td width="511" valign="top" style="width:383.25pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The OD<sub>600 </sub>of   the bacteria solution before breaking down.</span></p></td>
+
−
          </tr>
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            <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">v<sub>reaction</sub></span></p></td>
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            <td width="511" valign="top" style="width:383.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The change of light    absorption value of crude enzyme solution at 620/590/615 nm) after reacting    with different experimental glutamic acid decarboxylase solution for 1min</span></p></td>
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            <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">v<sub>blank</sub></span></p></td>
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            <td width="511" valign="top" style="width:383.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The change of light absorption    value of crude enzyme solution at 620/590/615 nm) after reacting with    different blank glutamic acid decarboxylase solution for 1min</span></p></td>
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            <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">k<sub>reaction</sub></span></p></td>
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            <td width="511" valign="top" style="width:383.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(In the measurement    of standard curves of different experimental glutamic acid decarboxylase    reaction solution) The change of light absorption value at 620/590/615 nm    when NaOH standard solution consumed 1 nmol H<sup>+</sup></span></p></td>
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            <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">k<sub>blank</sub></span></p></td>
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            <td width="511" valign="top" style="width:383.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(In the measurement    of standard curves of different blank glutamic acid decarboxylase reaction    solution) The change of light absorption value at 620/590/615 nm when NaOH    standard solution consumed 1 nmol H<sup>+</sup></span></p></td>
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        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(4) </span></strong><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">γ-aminobutyrate (GABA) Release Test </span></strong></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; Pick  monoclones from LB plate and inoculate in 2 mL LBG liquid medium (pH 7.0)  containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and  220 rpm.</span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; Transfer the  solution to fresh LBG liquid medium (pH4.5) by 1:100. Culture the cells until  OD<sub>600</sub> is between 0.5 and 0.6 (to logarithmic phase) or culture for  about 16h directly to stationary phase and measure the  accurate OD<sub>600 </sub>(h</span><span style="font-family:'微软雅黑',sans-serif; ">ereinafter </span><span style="font-family:'微软雅黑',sans-serif; ">denoted as OD).</span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; For cells in  logarithmic phase, transfer 4OD bacterial solution to a 15 mL BD tube and  centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 3500rpm for 10 min. For cells  in stationary phase, transfer 4OD bacterial solution to a 1.5 mL EP tube and  centrifuge at 4℃ and 8000rpm for 2 min. Discard the  supernatant. Suspend the cells with 1mL cool 10mM sodium glutamate citric acid  buffer. Centrifuge at 4℃ and 12000rpm for 2 min.  Discard the supernatant with a pipette. Centrifuge again for 30s and remove the  supernatant completely with a pipette. Resuspend the cells to 4 OD/mL with cool  citric acid buffer containing 10mM monosodium glutamate. Perform the whole cell  reaction at 37℃ for 1 h in order that the bacteria  release sufficient GABA.</span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Centrifuge  at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 8000rpm for 2 min. Take 10μL supernatant and add to 90μL GABase  reaction buffer. React at 37℃. Measure the final OD<sub>600</sub>.</span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Draw the  standard growth curves: prepare standard solutions of 0 mM, 0.05 mM, 0.1 mM,  0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 2 mM and 4 mM. Measure the light  absorption at 340 nm wave after reacting for 1 h. Do three series of parallel  experiments.</span></p>
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        <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(5) NH<sub>4</sub><sup>+</sup> Release Test</span></strong></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;1&gt; For  stationary phase: pick monoclones from LB plate and inoculate in 2 mL LB-2%  (w/v) glucose liquid medium containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm. </span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">For logarithmic phase:  pick monoclones from LB plate and inoculate in 5 mL LB-2% (w/v) glucose liquid  medium containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm.</span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;2&gt; For  stationary phase: Prepare pH standard solution (pH 4.01,  7.00, 9.21) and balance them at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Balance LBG liquid medium (pH 4.5) at room  temperature. Measure OD<sub>600</sub>s of overnight-cultured bacteria solution  and the bacteria solution diluted 10 times. Calculate the OD<sub>600</sub> value of 10 times diluted solution with the formula in test (1). Add  corresponding amounts of bacteria solution to 20 mL LBG liquid medium (pH 4.5)  to make the initial OD<sub>600</sub> value 0.05. </span><span style="font-family:'微软雅黑',sans-serif; ">Transfer the solution to 10 mL LBG liquid medium (pH  4.5) containing antibiotic. Incubate at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm until OD<sub>600 </sub>value is between 0.5 and 0.6. </span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">For logarithmic phase:  Prepare pH standard solution (pH 4.01, 7.00, 9.21) and balance them at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Balance LBG liquid medium (pH 4.5) at room  temperature. Measure OD<sub>600</sub> of overnight-cultured bacteria solution.  Add 50mL LBG liqiud medium (pH 4.5) to the 1000mL empty conical flask and  antibiotic subsequently. Calculate the OD<sub>600</sub> value of 10 times  diluted solution with the formula in test (1). Add corresponding amounts of  bacteria solution to 20 mL LBG liquid medium (pH 4.5) to make the initial OD<sub>600</sub> value 0.05. Culture at 37℃ and 220rpm for 2-3 hours.  Stop the growth with ice bath when OD<sub>600</sub> value is between 0.5 and  0.6.</span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;3&gt; Place the  cells on ice for at least 10 min to cool down. Transfer 20OD<sub>600</sub> bacterium solution to a 50 mL centrifuge tube. Place back on ice to inhibit  growth. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and  3500rpm for 10 min. Discard the supernatant. Centrifuge again for 2 min and  remove the supernatant completely with a pipette. Suspend the cells with 10 mL  cool sodium citrate solution. Centrifuge at 4℃ and  3500rpm for 10 min. Place the cells on ice.</span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;4&gt; Culture the  cells in the LBG liquid medium (pH 4.5) to logarithmic phase. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 3500rpm for 10 min. Resuspend the cells with 20 mL  25 mM sodium citrate solution containing 10 mM glutamine (pH 4.5) to a  concentration of 10OD<sub>600</sub>/mL.</span></p>
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        <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">&lt;5&gt; Use a  DX218-NH<sub>4</sub><sup>+ </sup>ion selective electrode to continuously  measure changes of NH<sub>4</sub><sup>+</sup> concentration in the buffer for  30min with a time interval of 1 min.</span></p>
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  <section class="popout protocols">
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    <div class="collapsible"> Sequencing Results Analysis</div>
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      <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The software MEGA 7 is  used to align sequences.</span></p>
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Latest revision as of 02:11, 22 October 2019

Ruby - Responsive Corporate Tempalte


Our team have seven wet lab experimenters, all of whom are laboratory rookies at first. We still remembered how hard for us to spend about 3 months completing the first plasmid construction. After one year iGEM training, we are skilled in molecular cloning operation - can complete multiple plasmids construction simultaneously in 3 days at the soonest now. During this time, many experiments failures we have experienced. But we have got a lot of joy from the success of the experiments at the same time. Through the training for the whole project, we deeply understand the importance of laboratory safety and strict implementation of protocols when performing an experiment. Consequently, we would like to post our protocols detailed enough for other team to repeat our experiments. Simply click on a protocol to read its steps. The side menu to scroll to the specific protocol is convenient for you to look for.

Modification of chassis
Verification of acid tolerant factors
Verification of Toehold Switches
Improvement of T7 promoters
Construction of VerProS pool
Construction of working part
Test of VerProS system