Difference between revisions of "Team:Kyoto/Protocol"

 
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<body>
 
<body>
 +
div class="clear"></div>
 
<div id="jump">
 
<div id="jump">
−
<a href="#wrapper"><img src="https://static.igem.org/mediawiki/2018/b/b5/T--Kyoto--UpButton.png"></a></div>
 
−
  <div id="BACKGROUND">
 
−
  <div id="wrapper"><br><br><br>
 
  
−
  <h1>Protocol(未)</h1><br><p>締め切り：　、原稿担当：</p>
+
<a href="#wrapper">
 +
<img src="https://static.igem.org/mediawiki/2018/1/11/T--Kyoto--upbotton.jpg"></a></div>
 +
<div id="BACKGROUND">
  
−
    <h5>Table</h5>
+
 
 +
<div style='padding-top: 100px;'><h1 id="wrapper"><img src="https://static.igem.org/mediawiki/2018/8/8b/T--Kyoto--protocol.png" width="30%"></div></h1>
 +
 
 +
<ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
 +
"><font face="Segoe UI">
 +
<b>Table</b></font></ol>
 
<div class="bg-primary">
 
<div class="bg-primary">
 
<section>
 
<section>
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                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P1-collapse" aria-expanded="false" aria-controls="P1-collapse">
 
                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P1-collapse" aria-expanded="false" aria-controls="P1-collapse">
 
<div>
 
<div>
−
                    <div class="col-md-11">Chemical Transformation</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+
 
 +
 
 +
      <ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
 +
"><font face="Segoe UI">
 +
Chemical Transformation</font></ol>
 +
<div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
</div>
 
</div>
 
                     </a>
 
                     </a>
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<p>
 
<p>
 
<b>Materials:</b><br>
 
<b>Materials:</b><br>
 +
Competent E.coli(Dh5alpha)
 
LB broth<br>
 
LB broth<br>
 
Ice<br>
 
Ice<br>
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<br>
 
<br>
 
<b>Methods:</b><br>
 
<b>Methods:</b><br>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
−
<li>Thaw 50µL competent E. coli cells on ice for 10 minutes<br></li>
+
<li>Thaw 10µL competent E. coli cells on ice for 10 minutes<br></li>
 
<li>Add:
 
<li>Add:
 
<ul style="font-size:16px;">
 
<ul style="font-size:16px;">
−
  <li>5-10 µl DNA from a ligation reaction mix or </li>
+
  <li>-2.5 µl DNA from a ligation reaction mix or </li>
−
<li>10-100ng DNA of a known plasmid </li>
+
<li> DNA of a known plasmid </li>
 
</ul>
 
</ul>
 
</li>
 
</li>
 
<li>Carefully flick the tube 4-5 times to mix cells and DNA. <b>Do not vortex.</b></li>
 
<li>Carefully flick the tube 4-5 times to mix cells and DNA. <b>Do not vortex.</b></li>
−
<li>Place the mixture on ice for 30 minutes. <b>Do not mix.</b></li>
+
<li>Place the mixture on ice for 15 minutes. <b>Do not mix.</b></li>
−
<li>Heat shock at exactly 42°C for exactly 30 seconds. <b>Do not mix.</b></li>
+
<li>Heat shock at exactly 42°C for exactly 45 seconds. <b>Do not mix.</b></li>
−
<li>Place on ice for 5 minutes. <b>Do not mix.</b></li>
+
<li>Place on ice for 1-3 minutes. <b>Do not mix.</b></li>
−
<li>Pipette 950 µl of room temperature SOC or LB media into the mixture.</li>
+
<li>Pipette 100 µl of room temperature SOC or LB media into the mixture.</li>
−
<li>Incubate at 37°C and 200-250 rpm for 60 minutes.</li>
+
<li>Incubate at 37°C for 40 minutes.</li>
 
<li>Mix the cells thoroughly by flicking the tube and inverting.</li>
 
<li>Mix the cells thoroughly by flicking the tube and inverting.</li>
 
<li>Spread:
 
<li>Spread:
 
<ul style="font-size:16px;">
 
<ul style="font-size:16px;">
−
<li>For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
+
<li>All of each transformation reaction onto a selection plate. If the reaction is too much　:
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
 
<li>Pellet cells at 8000rpm for 3 minutes</li>
 
<li>Pellet cells at 8000rpm for 3 minutes</li>
−
<li>Remove and dispense 600 µL of supernatant </li>
+
<li>Remove and dispense appropriate volume of supernatant </li>
 
<li>Re-suspend cells by light vortexing</li>
 
<li>Re-suspend cells by light vortexing</li>
 
<li>Plate resuspended cells as above</li>
 
<li>Plate resuspended cells as above</li>
 
</ol></li>
 
</ol></li>
−
 
−
<li>For known plasmid: 10 & 100 µL of each transformation reaction onto a selection plate. For the rest of 890 µL:
 
−
<ol style="font-size:16px;">
 
−
<li>Pellet cells at 8000rpm for 3 minutes</li>
 
−
<li>Remove and dispense 600 µL of supernatant </li>
 
−
<li>Re-suspend cells by light vortexing</li>
 
−
<li>Plate resuspended cells as above</li>
 
−
</ol></li>
 
−
</ul>
 
−
<li>Incubate overnight at 37°C with plates upside down.</li>
 
−
</ol>
 
  
 
</p>
 
</p>
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                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P2-collapse" aria-expanded="false" aria-controls="P2-collapse">
 
                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P2-collapse" aria-expanded="false" aria-controls="P2-collapse">
 
<div>
 
<div>
−
                    <div class="col-md-11">Growing Overnight Cultures</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+
<ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
 +
"><font face="Segoe UI">
 +
Growing Overnight Cultures</font></ol>
 +
                    <div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
</div>
 
</div>
 
                     </a>
 
                     </a>
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<p>
 
<p>
 
<b>Materials:</b><br>
 
<b>Materials:</b><br>
−
5 ml LB broth<br>
+
3 ml LB broth<br>
−
5 μl antibiotic<br>
+
3 μl antibiotic<br>
−
Loops<br>
+
Tips<br>
−
12 ml culture tube<br>
+
culture tube<br>
 
<br>
 
<br>
 
<b>Methods:</b><br>
 
<b>Methods:</b><br>
−
Overnight cultures were prepared under sterile conditions using a Bunsen burner<br>
+
Overnight cultures were prepared under sterile conditions using a clean bench<br>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
−
<li>Add 5 ml liquid LB media into 12 ml culture tubes</li>
+
<li>Add 3 ml liquid LB media into 12 ml culture tubes</li>
−
<li>Add 5 μl of appropriate antibiotic into the broth</li>
+
<li>Add 3 μl of appropriate antibiotic into the broth</li>
−
<li>Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth</li>
+
<li>Using the tip, pick a single colony and inoculate the cultures by dipping the loop into the LB broth</li>
−
<li>Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm</li>
+
<li>Seal the tubes and incubate overnight at 37°C shaking at 130-150 rpm</li>
 
</ol>
 
</ol>
 
</p>
 
</p>
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                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P3-collapse" aria-expanded="false" aria-controls="P3-collapse">
 
                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P3-collapse" aria-expanded="false" aria-controls="P3-collapse">
 
<div>
 
<div>
−
                    <div class="col-md-11">PCR From Plasmid DNA Template</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+
<ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
 +
"><font face="Segoe UI">
 +
PCR From Plasmid DNA Template</font></ol>
 +
<div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
</div>
 
</div>
 
                     </a>
 
                     </a>
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                     <div class="panel-body">
 
                     <div class="panel-body">
 
<p>
 
<p>
−
<b>Materials:</b><br>
+
<b>Materials:   We used various PCR Polymerase, and we obeyed each protocols. We show our most frequent protocol here.</b><br>
−
2x Phusion Mastermix<br>
+
2x Q5 Mastermix<br>
−
10 µM forward primer<br>
+
 
10 µM forward primer<br>
 
10 µM forward primer<br>
 +
10 µM reverse primer<br>
 
PCR tube<br>
 
PCR tube<br>
 
Sterile water<br>
 
Sterile water<br>
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<br>
 
<br>
 
<b>Methods:</b><br>
 
<b>Methods:</b><br>
−
For a 25 µL reaction<br>
 
−
<ol style="font-size:16px;">
 
−
<li>In a PCR tube on ice, combine  1-10 ng of plasmid DNA, 1.25 µL of 10 µM forward primer, 1.25 µL of 10 µM reverse primer to a PCR tube on ice, 12.5 µL of 2x Phusion Mastermix, and sterile water up to 25 µL.
 
−
<br><i>Note: It is important to add Phusion Master Mix last in order to prevent primer degradation
 
−
caused by the 3 ́→ 5 ́ exonuclease activity</i></li>
 
−
<li>Gently mix the reaction</li>
 
−
<li>If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly</li>
 
−
<li>Transfer the PCR tube from ice to a PCR machine to begin thermocycling</li>
 
−
</ol>
 
  
 
<p><br>For a 50 µL reaction<br></p>
 
<p><br>For a 50 µL reaction<br></p>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
−
<li>In a PCR tube on ice, combine  1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phusion Mastermix, and sterile water up to 50 µL.
+
<li>In a PCR tube on ice, combine  1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Q5 Mastermix, and sterile water up to 50 µL.
−
<br><i>Note: It is important to add Phusion Master Mix last in order to prevent primer degradation  
+
<br><i>Note: It is important to add Q5 Master Mix last in order to prevent primer degradation  
 
caused by the 3 ́→ 5 ́ exonuclease activity</i></li>
 
caused by the 3 ́→ 5 ́ exonuclease activity</i></li>
 
<li>Gently mix the reaction</li>
 
<li>Gently mix the reaction</li>
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</table>
 
</table>
  
−
<p id="Note1">Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Phusion: <a target="_blank" href="http://tmcalculator.neb.com/#!/">http://tmcalculator.neb.com/#!/</a></p>
+
<p id="Note1">Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Q5: <a target="_blank" href="http://tmcalculator.neb.com/#!/">http://tmcalculator.neb.com/#!/</a></p>
  
  
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</div>
 
</div>
 
</div>
 
</div>
−
 
 
<div class="some-padding"></div>
 
<div class="some-padding"></div>
 
<div class="some-padding"></div>
 
<div class="some-padding"></div>
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<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
 
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
 
             <div class="panel panel-default">
 
             <div class="panel panel-default">
−
                 <div class="panel-heading" role="tab" id="P4">
+
                 <div class="panel-heading" role="tab" id="P3">
 
                     <h4 class="panel-title">
 
                     <h4 class="panel-title">
−
                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P4-collapse" aria-expanded="false" aria-controls="P4-collapse">
+
                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P3-collapse" aria-expanded="false" aria-controls="P3-collapse">
 
<div>
 
<div>
−
                    <div class="col-md-11">Colony PCR</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+
<ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
−
</div>                  
+
"><font face="Segoe UI">
 +
Colony PCR </font></ol>
 +
<div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 +
</div>
 
                     </a>
 
                     </a>
 
                     </h4>
 
                     </h4>
  
 
                 </div>
 
                 </div>
−
                 <div id="P4-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P4">
+
                 <div id="P3-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P3">
 
                     <div class="panel-body">
 
                     <div class="panel-body">
 +
 
<p>
 
<p>
 
<b>Materials:</b><br>
 
<b>Materials:</b><br>
 
Sterile Water<br>
 
Sterile Water<br>
−
25 µL RedTaq mastermix<br>
+
2x EmeraldAmp MAX mastermix<br>
−
1 E. coli colony<br>
+
E. coli colony<br>
−
2.5 µL of 10 µM forward primer<br>
+
10 µM forward primer<br>
−
2.5 µL of 10 µM reverse primer<br>
+
10 µM reverse primer<br>
 
<br>
 
<br>
 
<b>Methods:</b><br>
 
<b>Methods:</b><br>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
−
<li>Add a single colony of cells to 50 µL of water. Incubate at 95C for a minute to lyse the cells.</li>
+
<li>Make mastermix of reaction.Add 2.5µL of EmeraldAmp MAX,0.05µL of 10µM forward primer, and 0.05µL of 10µM reverse primer to 2.4µL of water each reaction.</li>
−
<li>Combine 1 µL cell lysate, 25 µL RedTaq mastermix, 2.5 µL of 10 µM forward primer, 2.5 µL of 10 µM reverse primer, and sterile water up to 50 µL. <br>
+
<li>Touch your objective colony with tip then touch the 5µL of reaction liquid in PCR tube.</li>
−
<i>Note: It is important to add RedTaq Master Mix last in order to prevent primer
+
<li>If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly</li>
−
degradation caused by the 3 ́→ 5 ́ exonuclease activity</i></li>
+
−
<li>Incubate in the thermocycler -  Taq has a lower optimum temperature than Phusion.</li>
+
  
 
</ol>
 
</ol>
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     </tbody>
 
     </tbody>
 
</table>
 
</table>
−
<p>Note: If loading on a gel, the RedTaq mix contains loading dye, so don’t add anything else.</p>  
+
<p>Note: If loading on a gel, the EmeraldAmp MAX mix contains loading dye, so don’t add anything else.</p>  
  
 
   </div>
 
   </div>
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<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
 
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
 
             <div class="panel panel-default">
 
             <div class="panel panel-default">
−
                 <div class="panel-heading" role="tab" id="P5">
+
                 <div class="panel-heading" role="tab" id="P3">
 
                     <h4 class="panel-title">
 
                     <h4 class="panel-title">
−
                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P5-collapse" aria-expanded="false" aria-controls="P5-collapse">
+
                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P3-collapse" aria-expanded="false" aria-controls="P3-collapse">
 
<div>
 
<div>
−
                    <div class="col-md-11">Preparation of LB Broth, Agar, and Glycerol Stocks</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+
<ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
−
</div>                  
+
"><font face="Segoe UI">
 +
Preparation of LB broth, Agar, Glycerol Stocks </font></ol>
 +
<div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 +
</div>
 
                     </a>
 
                     </a>
 
                     </h4>
 
                     </h4>
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<specialh3>LB Broth</specialh3><br>
 
<specialh3>LB Broth</specialh3><br>
 
<b>Materials:</b><br>
 
<b>Materials:</b><br>
−
25 g LB broth (powder)<br>
+
10 g Tripton<br>
−
1 Litre Purified Water<br>
+
5g Yeast Extract<br>
 +
5g NaCl<br>
 +
1L Purified Water<br>
 
<br>
 
<br>
 
<b>Methods:</b><br>
 
<b>Methods:</b><br>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
−
<li>Add 25g LB broth to 1 litre purified water</li>
+
<li>Add these reagents to 1L purified water</li>
 
<li>Autoclave</li>
 
<li>Autoclave</li>
 
</ol>
 
</ol>
 
</p>
 
</p>
−
 
+
<br>
 
<p>
 
<p>
 
<specialh3>LB Agar</specialh3><br>
 
<specialh3>LB Agar</specialh3><br>
 
<b>Materials:</b><br>
 
<b>Materials:</b><br>
−
37 g LB Agar (powder)<br>
+
10 g Tripton<br>
−
1 Litre Purified Water<br>
+
5g Yeast Extract<br>
 +
5g NaCl<br>
 +
15g Agar<br>
 +
1L Purified Water<br>
 
<br>
 
<br>
 
<b>Methods:</b><br>
 
<b>Methods:</b><br>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
−
<li>Add 37g LB Agar to 1 litre purified water</li>
+
<li>Add these reagent to 1 L purified water</li>
 
<li>Autoclave</li>
 
<li>Autoclave</li>
 
</ol>
 
</ol>
 
</p>
 
</p>
−
 
+
<br>
 
<p>
 
<p>
 
<specialh3>Glycerol Stocks</specialh3><br>
 
<specialh3>Glycerol Stocks</specialh3><br>
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<br>
 
<br>
 
<b>Methods:</b><br>
 
<b>Methods:</b><br>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
 
<li>Add 500µl glycerol (80%) to 1.5ml eppendorf tube</li>
 
<li>Add 500µl glycerol (80%) to 1.5ml eppendorf tube</li>
 
<li>Add 500µl overnight culture in LB</li>
 
<li>Add 500µl overnight culture in LB</li>
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<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
 
             <div class="panel panel-default">
 
             <div class="panel panel-default">
−
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+
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                     <h4 class="panel-title">
 
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−
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+
                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P3-collapse" aria-expanded="false" aria-controls="P3-collapse">
 
<div>
 
<div>
−
                    <div class="col-md-11">DNA Gel Electrophoresis</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+
<ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
 +
"><font face="Segoe UI">
 +
DNA Electrophoresis </font></ol>
 +
<div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
</div>
 
</div>
 
                     </a>
 
                     </a>
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TAE buffer<br>
 
TAE buffer<br>
 
Gel mould<br>
 
Gel mould<br>
−
5-10 µL SybrSafe<br>
+
2-4µL EtBr Solution(10mg/ml)<br>
 
Gel Tank<br>
 
Gel Tank<br>
−
8-10 µL DNA ladder<br>
+
5-7 µL DNA ladder<br>
 
DNA loading dye<br>
 
DNA loading dye<br>
 
<br>
 
<br>
 
<b>Methods:</b><br>
 
<b>Methods:</b><br>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
−
<li>Prepare 8% w/v solution of agarose powder in 1/10 TAE buffer (e.g. 0.8g agarose powder in 100 mL buffer) using a conical flask</li>
+
<li>Prepare 1% w/v solution of agarose powder in 1x TAE buffer (e.g. 1g agarose powder in 100 mL buffer) using a conical flask</li>
 
<li>Heat the mixture until agarose is completely dissolved. Do not let the solution boil.</li>
 
<li>Heat the mixture until agarose is completely dissolved. Do not let the solution boil.</li>
 
<li>Pour the solution into a gel mould</li>
 
<li>Pour the solution into a gel mould</li>
−
<li>Add 5-10 µL SybrSafe® to the solution. Make sure there are no bubbles in the solution.</li>
+
<li>Add 2-4 µL EtBr Solution (10mg/ml) to the solution. Make sure there are no bubbles in the solution.</li>
−
<li>Allows the solution to set (approx 15-20 minutes)</li>
+
<li>Allows the solution to set (approx 20-30 minutes)</li>
 
<li>Transfer the agarose gel to a tank, remove the comb and apply:
 
<li>Transfer the agarose gel to a tank, remove the comb and apply:
 
<ul>
 
<ul>
−
<li>8-10 µL of the DNA ladder </li>
+
<li>5-7 µL of the DNA ladder </li>
−
<li>DNA samples with the corresponding amount of DNA loading dye (6X)</li>
+
<li>DNA samples with the corresponding amount of DNA loading dye (10X)</li>
 
</ul></li>
 
</ul></li>
−
<li>Run the gel for 45-60 minutes at 100V</li>
+
<li>Run the gel for 25-35 minutes at 100V</li>
  
 
</ol>
 
</ol>
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+
<div>
−
                    <div class="col-md-11">Ligation</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+
<ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
 +
"><font face="Segoe UI">
 +
Ligation </font></ol>
 +
<div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
</div>
 
</div>
 
                     </a>
 
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Microcentrifuge tube<br>
 
Microcentrifuge tube<br>
 
Ice<br>
 
Ice<br>
−
1 µL T4 DNA Ligase<br>
+
2 µL 2X Ligationhigh<br>
−
2 µL 10X T4 DNA ligase buffer<br>
+
Vector Plasmid solution<br>
−
50ng Vector Plasmid<br>
+
Insert DNA solution<br>
−
Insert DNA<br>
+
−
Sterile water<br>
+
 
<br>
 
<br>
 
<b>Methods:</b><br>
 
<b>Methods:</b><br>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
−
<li>Calculate volumes of vector and insert DNA required using NEBioCalculator (<a href="http://nebiocalculator.neb.com/#!/ligation" > http://nebiocalculator.neb.com/#!/ligation</a> - required molar ratio of 1:3::vector:insert)</li>
+
<li>Calculate volumes of vector and insert DNA required using NEBioCalculator (required molar ratio of 1:3::vector:insert)</li>
−
<li>Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube on ice (add T4 DNA ligase last)</li>
+
<li>Add vector plasmid, insert DNA, and 2x Ligationhigh to the microcentrifuge tube on ice (add T4 DNA ligase last)</li>
−
<li>Make reaction up to 20 µL using sterile water</li>
+
<li>Incubate at 16℃ for 30 - 60 min for sticky ends or  2-16 hours for blunt ends</li>
−
<li>Incubate at room temperature for 30 - 60 min for sticky ends or  1-2 hours for blunt ends</li>
+
 
</ol>
 
</ol>
  
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                     <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P8-collapse" aria-expanded="false" aria-controls="P8-collapse">
 
<div>
 
<div>
−
                    <div class="col-md-11">Restriction Digestion (Test Digest and Assembly Digest)</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+
<ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
 +
"><font face="Segoe UI">
 +
Restriction Digestion (Test Digest and Assembly Digest)</font></ol>
 +
<div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
</div>                     
 
</div>                     
  
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     </tbody>
 
     </tbody>
 
</table>
 
</table>
−
<ol style="font-size:16px;">
+
<ol style="font-size:16px;margin-left:15%;">
 
<li>Set up the reaction following the instruction in the table above , depending on whether test digest or assembly digest is being performed.</li>
 
<li>Set up the reaction following the instruction in the table above , depending on whether test digest or assembly digest is being performed.</li>
 
<li> Incubate digestion reaction at 37°C:
 
<li> Incubate digestion reaction at 37°C:
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−
<div>
 
−
                    <div class="col-md-11">Growth Characterisation</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
−
</div>
 
−
                    </a>
 
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−
                    <div class="panel-body">
 
−
<p>
 
−
<b>Materials:</b><br>
 
−
Microcentrifuge tube<br>
 
−
Media of choice<br>
 
−
Overnight Culture of Cells<br>
 
−
96 well plate<br>
 
−
Plate reader<br>
 
−
<br>
 
−
<b>Methods:</b><br>
 
−
<ol style="font-size:16px;">
 
−
<li>Dilute overnight cultures to 0.1 OD. <b>Don’t forget the media control.</b></li>
 
−
<li>Seed your cells in the 96 well plate at 100μL per well.</li>
 
−
<li>Make reaction up to 20 µL using sterile water</li>
 
−
<li>Set the plate reader for 600OD and run it for 12 hours for bacteria or 24 hours plus for yeast
 
−
</li>
 
−
</ol>
 
−
 
−
  </div>
 
−
</div>
 
−
</div>
 
−
 
−
<div class="some-padding"></div>
 
−
<div class="some-padding"></div>
 
−
 
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−
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−
<div>
 
−
                    <div class="col-md-11">Cell Sorting for Co-Culture Characterisation</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
−
</div>
 
−
                    </a>
 
−
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−
 
−
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−
                    <div class="panel-body">
 
−
<p>
 
−
<b>Materials:</b><br>
 
−
Microcentrifuge tube<br>
 
−
Media of choice<br>
 
−
Overnight Culture of Cells<br>
 
−
96 well plate<br>
 
−
Flow Cytometer<br>
 
−
PBS<br>
 
−
<br>
 
−
<b>Methods:</b><br>
 
−
<ol style="font-size:16px;">
 
−
<li>Use Flowjo to predefine the gates for the Flow cytometer. This allows the cells within the co culture to be counted based on size.</li>
 
−
<li>Dilute the Cultures to 1 OD (don’t forget the media control) and culture together at desired inoculation ratio</li>
 
−
<li>Set up samples in triplicate form on a 96 well plate. Dilute 10 fold and 100 fold in PBS.</li>
 
−
<li>Incubate Cells and take samples at desired time points to obtain growth curves</li>
 
−
</ol>
 
−
 
−
  </div>
 
−
</div>
 
−
</div>
 
−
 
−
<div class="some-padding"></div>
 
−
<div class="some-padding"></div>
 
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            <div class="panel panel-default">
 
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−
                    <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P11-collapse" aria-expanded="false" aria-controls="P11-collapse">
 
−
 
−
<div>
 
−
                    <div class="col-md-11">Quorum Characterisation Experiments</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 
−
</div>                   
 
−
                    </a>
 
−
                    </h4>
 
−
 
−
                </div>
 
−
                <div id="P11-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P11">
 
−
                    <div class="panel-body">
 
−
<p>
 
−
<b>Materials:</b><br>
 
−
Replica plate or glycerol stock of cells<br>
 
−
Appropriate antibiotic<br>
 
−
LB broth<br>
 
−
96 well plate<br>
 
−
AHLs (at concentrations of 10pM, 100pM, 1nM, 10nM, 100nM, 1µM, 10µM and 100µM)<br>
 
−
<br>
 
−
<b>Methods:</b><br>
 
−
<ol style="font-size:16px;">
 
−
<li>Grow overnight culture of cells, either from replica plate or glycerol stock (see overnight culture protocol)
 
−
</li>
 
−
<li>In the morning, add 100 µL of the cell suspension to 10 mL LB and 10µL of antibiotic</li>
 
−
<li>Grow cells in incubator for 3-4 hours at 200rpm and 37°C</li>
 
−
<li>Blank spectrophotometer with 1mL LB and measure OD of cells (dilute 10x in LB)</li>
 
−
<li>Make up suspension of cells at OD 0.05</li>
 
−
<li>Add 100µL of cells to each well in the 96-well plate using the block-filler machine</li>
 
−
<li>Using the multi-channel pipette add 2µL of intended AHL to each well, add samples in triplicate form
 
−
</li>
 
−
<li>Using the multi-channel pipette add 2µL of intended AHL to each well, add samples in triplicate form
 
−
</li>Run on plate-reader for 12 hours, with fluorescence and absorbance measurements taken every 15 minutes (set plate reader to 37°C and 500rpm shaking)
 
−
</li>
 
−
</ol>
 
−
 
−
  </div>
 
−
</div>
 
−
</div>
 
  
 
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<div class="some-padding"></div>
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  <div>
 
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                    <div class="col-md-11">PCR Purification, Gel Extraction & Miniprep</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
+
<ol style="font-size:25px;margin-left:10%;color:#757575;border-bottom:4px solid #757575;
 +
"><font face="Segoe UI">
 +
PCR Purification, Gel Extraction & Miniprep</font></ol>
 +
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                     <div class="panel-body">
 
<p>
 
<p>
−
<b>PCR purification</b> was performed according to the QIAquick PCR Purification Kit<br>
+
<b>PCR purification</b> was performed according to the FastGene™Gel/PCR Extraction Kit<br>
−
<b>Gel extraction</b> was performed according to the QIAquick Gel Extraction Kit<br>
+
<b>Gel extraction</b> was performed according to the FastGene™Gel/PCR Extraction Kit <br>
−
<b>Minipreps</b> were carried out according to the QIAprep Spin Miniprep Kit<br>
+
<b>Minipreps</b> were carried out according to the FastGene™Plasmid Mini Kit<br>
  
  

Latest revision as of 13:30, 17 October 2018

div class="clear">

    Table

On this page, the ecolibrium team would like to share with you the protocols that we have been using over the summer. This library of protocols has been developed alongside our supervisors for the purpose of this study. Here you can find the exact methods we use to generate our data and results. We share them in the interest of reproducibility.

Materials:
Competent E.coli(Dh5alpha) LB broth
Ice
Selection plates

Methods:

  1. Thaw 10µL competent E. coli cells on ice for 10 minutes
  2. Add:
    • -2.5 µl DNA from a ligation reaction mix or
    • DNA of a known plasmid
  3. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  4. Place the mixture on ice for 15 minutes. Do not mix.
  5. Heat shock at exactly 42°C for exactly 45 seconds. Do not mix.
  6. Place on ice for 1-3 minutes. Do not mix.
  7. Pipette 100 µl of room temperature SOC or LB media into the mixture.
  8. Incubate at 37°C for 40 minutes.
  9. Mix the cells thoroughly by flicking the tube and inverting.
  10. Spread:
    • All of each transformation reaction onto a selection plate. If the reaction is too much　:
      1. Pellet cells at 8000rpm for 3 minutes
      2. Remove and dispense appropriate volume of supernatant
      3. Re-suspend cells by light vortexing
      4. Plate resuspended cells as above

Materials:
3 ml LB broth
3 μl antibiotic
Tips
culture tube

Methods:
Overnight cultures were prepared under sterile conditions using a clean bench

  1. Add 3 ml liquid LB media into 12 ml culture tubes
  2. Add 3 μl of appropriate antibiotic into the broth
  3. Using the tip, pick a single colony and inoculate the cultures by dipping the loop into the LB broth
  4. Seal the tubes and incubate overnight at 37°C shaking at 130-150 rpm

Materials: We used various PCR Polymerase, and we obeyed each protocols. We show our most frequent protocol here.
2x Q5 Mastermix
10 µM forward primer
10 µM reverse primer
PCR tube
Sterile water
Plasmid DNA

Methods:


For a 50 µL reaction

  1. In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Q5 Mastermix, and sterile water up to 50 µL.
    Note: It is important to add Q5 Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
  2. Gently mix the reaction
  3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
  4. Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling


Thermocycling
The PCR machine should be set to run the following steps:

Step Temperature (°C) Time
Initial denaturation 98 30 seconds
25-35 cycles 98 (denaturation)
45-72 (annealing) see Note 1
72 (extension)		 5-10 seconds
10-30 seconds
15-30 seconds per kb
Final extension 72 2-5 minutes
Hold 4 Indefinitely

Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Q5: http://tmcalculator.neb.com/#!/

Materials:
Sterile Water
2x EmeraldAmp MAX mastermix
E. coli colony
10 µM forward primer
10 µM reverse primer

Methods:

  1. Make mastermix of reaction.Add 2.5µL of EmeraldAmp MAX,0.05µL of 10µM forward primer, and 0.05µL of 10µM reverse primer to 2.4µL of water each reaction.
  2. Touch your objective colony with tip then touch the 5µL of reaction liquid in PCR tube.
  3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly


Thermocycling
The PCR machine should be set to run the following steps:

Step Temperature (°C) Time
Initial denaturation 98 30 seconds
25-35 cycles 98 (denaturation)
45-72 (annealing) see Note 1
68 (extension)		 5-10 seconds
10-30 seconds
15-30 seconds per kb
Final extension 72 5-10 minutes
Hold 4 Indefinitely

Note: If loading on a gel, the EmeraldAmp MAX mix contains loading dye, so don’t add anything else.

LB Broth
Materials:
10 g Tripton
5g Yeast Extract
5g NaCl
1L Purified Water

Methods:

  1. Add these reagents to 1L purified water
  2. Autoclave


LB Agar
Materials:
10 g Tripton
5g Yeast Extract
5g NaCl
15g Agar
1L Purified Water

Methods:

  1. Add these reagent to 1 L purified water
  2. Autoclave


Glycerol Stocks
Materials:
500µl glycerol (80%)
500µl overnight culture in LB

Methods:

  1. Add 500µl glycerol (80%) to 1.5ml eppendorf tube
  2. Add 500µl overnight culture in LB
  3. Store at -80°C

Materials:
Agarose Powder
TAE buffer
Gel mould
2-4µL EtBr Solution(10mg/ml)
Gel Tank
5-7 µL DNA ladder
DNA loading dye

Methods:

  1. Prepare 1% w/v solution of agarose powder in 1x TAE buffer (e.g. 1g agarose powder in 100 mL buffer) using a conical flask
  2. Heat the mixture until agarose is completely dissolved. Do not let the solution boil.
  3. Pour the solution into a gel mould
  4. Add 2-4 µL EtBr Solution (10mg/ml) to the solution. Make sure there are no bubbles in the solution.
  5. Allows the solution to set (approx 20-30 minutes)
  6. Transfer the agarose gel to a tank, remove the comb and apply:
    • 5-7 µL of the DNA ladder
    • DNA samples with the corresponding amount of DNA loading dye (10X)
  7. Run the gel for 25-35 minutes at 100V

Materials:
Microcentrifuge tube
Ice
2 µL 2X Ligationhigh
Vector Plasmid solution
Insert DNA solution

Methods:

  1. Calculate volumes of vector and insert DNA required using NEBioCalculator (required molar ratio of 1:3::vector:insert)
  2. Add vector plasmid, insert DNA, and 2x Ligationhigh to the microcentrifuge tube on ice (add T4 DNA ligase last)
  3. Incubate at 16℃ for 30 - 60 min for sticky ends or 2-16 hours for blunt ends

Materials:
Restriction Enzyme: NEB enzyme finder used to see which restriction enzymes are required
10X buffer: NEB double digest finder used to see which buffers are required for the particular restriction enzymes
Plasmid DNA
Sterile water

Methods:

Component Test digest
Double digestion (20 µL, for construct analysis) 		Assemble digest
Double digestion (20-50 µL, for gene assembly)
Sterile water to 20 µL to 50 µL
10X buffer 2 µL 2-5 µL
Plasmid DNA ~200 ng for test digest, (DNA mass is variable dependent on insert size.
Smallest digestion fragment mass should be > 50ng)		 1,000 -2,000 ng
Restriction enzyme 0.5 µL + 0.5 µL 1 µL + 1 µL
  1. Set up the reaction following the instruction in the table above , depending on whether test digest or assembly digest is being performed.
  2. Incubate digestion reaction at 37°C:
    1. 30 min for Test digest
    2. 2-3 hours or overnight for Assembly digest
  3. Perform heat deactivation at 80°C for 20 minutes, if not running on a gel at the end of incubation.

PCR purification was performed according to the FastGene™Gel/PCR Extraction Kit
Gel extraction was performed according to the FastGene™Gel/PCR Extraction Kit
Minipreps were carried out according to the FastGene™Plasmid Mini Kit