Difference between revisions of "Team:Macquarie Australia/Experiments"

 
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{{Macquarie_Australia}}
 
 
{{Macquarie_Australia/Nav2Project}}
 
{{Macquarie_Australia/Nav2Project}}
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{{Macquarie_Australia}}
 
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<title>Experiments &amp; Protocols</title>
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<title>Experiments and Protocol</title>
 
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<div class="centreStuffInline">
 
<div class="centreStuffInline">
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<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/ProjectOverview"><img src="https://static.igem.org/mediawiki/2015/a/a0/MqAust_1Project_v06a-150dpi.png" width="220px" alt="Link to Project page"></a></figure>
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<img src="https://static.igem.org/mediawiki/2015/0/07/NDproj2test.jpeg">
 
</div>
 
</div>
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<div class="centreStuffInline">
 
<div class="centreStuffInline">
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<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Description"><img src="https://static.igem.org/mediawiki/2015/e/ed/MqAust_BubbleProject_1Description.png" width="110px" alt="Link to Project Description page"></a></figure>
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<figure class="specialInline"><img src="https://static.igem.org/mediawiki/2015/e/ed/MqAust_BubbleProject_1Description.png" width="110px" alt="Project Description page"></figure>
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<figure class="specialInline"><img src="https://static.igem.org/mediawiki/2015/9/91/MqAust_BubbleProject_2Experiments.png" width="110px" alt="Experiments &amp; Protocols page"></figure>
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<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Experiments"><img src="https://static.igem.org/mediawiki/2015/9/91/MqAust_BubbleProject_2Experiments.png" width="110px" alt="Link to Experiments &amp; Protocols page"></a></figure>
 
<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Results"><img src="https://static.igem.org/mediawiki/2015/0/08/MqAust_BubbleProject_3Results.png" width="110px" alt="Link to Results page"></a></figure>
 
<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Results"><img src="https://static.igem.org/mediawiki/2015/0/08/MqAust_BubbleProject_3Results.png" width="110px" alt="Link to Results page"></a></figure>
 
<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Notebook"><img src="https://static.igem.org/mediawiki/2015/9/98/MqAust_BubbleProject_5Notebook.png" width="110px" alt="Link to Notebook page"></a></figure>
 
<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Notebook"><img src="https://static.igem.org/mediawiki/2015/9/98/MqAust_BubbleProject_5Notebook.png" width="110px" alt="Link to Notebook page"></a></figure>
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</div>
 
</div>
  
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<h2 style="text-align: center;"> Experiments &amp; Protocols</h2>
 
  
 +
<br>
 +
<center><img src="https://static.igem.org/mediawiki/2015/3/39/NDprotocolflow.jpeg"></center>
 +
<figcaption><center><i><b>Fig 1.</b> An overview of the methodology. A modified BioBrick Standard Assembly was used. Alkaline phosphatase on the target vector backbone for the genes; ChlM and those from Photosystem II. The Gibson Assembly method was used in order to insert composite part ChlH into the BioBrick vector plasmid.</i></figcaption></center></br>
  
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      <div class="centreStuffInline">
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    <h1 style="text-align: center;">Experiments and Protocol</h1>
 +
    <h4 style="text-align: center;">Making Competent Cells</h4>
 +
<ol>
 +
<li>Using a sterile plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22<sup>o</sup>C, 200-250rpm.</li>
 +
<li>A600 should be 0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5.</li>
 +
<li>Remove the flask from the incubator and place on ice for 10 minutes.</li>
 +
<li>Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4<sup>o</sup>C</li>
 +
<li>Pour off and discard the supernatant, and immediately place the tube on ice.</li>
 +
<li>Resuspend your cells in 1mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.</li>
 +
<li>Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30mL in this case). Mix the tube by gently inverting 3 times.</li>
 +
<li>Incubate the tube on ice for 10 minutes.</li>
 +
<li>Centrifuge at 2,500 x g for 7 minutes at 4<sup>o</sup>C, discard the supernatant.</li>
 +
<li>Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. e.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.</li>
 +
</li>Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.</li>
 +
<li>Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.</li>
 +
<li>Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen.</li>
 +
<li>Repeat step 11-13 for the rest of your cell suspension in step 10. Store cells at -80<sup>o</sup>C</li>
 +
</ol>
 +
<h6>TB Buffer</h6>
 +
<b>Ingredients:</b> 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl.<br>
 +
<b>Methods:</b> All components (except for MnCl2-4H2O) were mixed and dissolved in 500 mL of water and pH adjusted to 6.7 with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL of water, mixed and solution adjusted to 1 L. Sterilisation via filtration followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.
 +
<h6>SOB Media (for competent cells)</h6>
 +
<b>Ingredients:</b> 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl NaCl, 417µL KCl, 1.205g Mg2SO4 & 500mL Milli-Q water. <br>
 +
<b>Methods:</b> The adjusted quantities were combined in a 1 L measuring column with constant stirring and then placed in the autoclave for sterilisation (121<sup>o</sup>C, 15 min, standard liquid cycle).<br><br>
  
−
      <img src="https://static.igem.org/mediawiki/2015/a/ab/MQAust_OverView_FlowChart.png">
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    <h4 style="text-align: center;"> Agarose Gel Electrophoresis</h4>
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      <div class="centreStuffInline"><i><b>Figure 1.</b> An overview of the methodology. </i> A modified BioBrick Standard Assembly was used. Alkaline phosphatase (referenced below) on the target vector backbone for the genes; ChlM and those from Photosystem II. The Gibson Assembly method was used in order to insert composite part ChlH into the BioBrick vector plasmid (referenced below). </br></p>
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<ol>
 +
<li>Mix 1ul of 1kbp DNA ladder with 6ul of loading dye (bromophenol blue) and 4ul of 1x TAE buffer and load into first well </li>
 +
<li>Mix 5ul of plasmid DNA with 1ul of loading dye and load into wells.</li>
 +
<li>Run gel at 90V for 45minutes approximately</li>
 +
<li>Gel image is taken using Biorad GelDoc</li>
 +
</ol>
 +
<h6>EDTA Buffer</h6>
 +
<b>Ingredients:</b> 37.22g EDTA solid, 180 mL of water and pH adjusted to 8.0 using 10M NaOH.<br>
 +
<b>Methods:</b> Components were combined then pH adjusted.
 +
<h6>50 X TAE Buffer</h6>
 +
<b>Ingredients:</b> 121.2g Tris base (dissolved in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0).<br>
 +
<b>Methods:</b> Components were combined then make up to 500ml with distilled water.
 +
<h6>Making Agarose Gel</h6>
 +
<b>Ingredients:</b> 1g agarose powder, 100ml of 1 x TAE<br>
 +
<b>Methods:</b> Dissolve agarose in TAE by microwaving for 1 minute, wait till cool to touch, then add 2ul of GelRed and pour into the casette.
 +
<br></br>
  
−
      </div>
+
    <h4 style="text-align: center;">Composite Part Ligation</h4>
−
<p>This page is for descriptions of the experiments, research and protocols we used in our iGEM project.</p>
+
<p>The assembly of composite parts from two existing BioBricks i.e. BioBrick plasmid A (parent vector) and BioBrick plasmid B (gene to be inserted) was performed through a restriction digest/ligation protocol. </p>
 +
<ol>
 +
<li>200ng of each BioBrick plasmid was digested as follows: Plasmid BioBrick A with NEB restriction enzymes SpeI and PstI; plasmid BioBrick B with XbaI and PstI, according to supplier protocol. (1hr @37<sup>o</sup>C, then 20mins @80<sup>o</sup>C).</li>
 +
<li>1µL of Fast alkaline phosphatase (Thermo Scientific) was added to reaction tube of plasmid BioBrick A to dephosphorylate the gene fragments and prevent re-ligation of the parent vector. Reaction tubes were incubated at 37<sup>o</sup>C for 60 mins, followed by a deactivation step at 80<sup>o</sup>C for 20 mins.</li>
 +
<li>Ligation was then performed with an insert to vector molar ratio of 3:1. 1µL of T4 ligase (NEB) was added to the mix for ligation, according to supplier protocol. Ligation reactions were performed at 37<sup>o</sup>C for 60 mins and kept on ice for transformation into chemical competent cells.</li>
 +
</ol>
 +
<br></br>
  
−
<h5>What should this page contain?</h5>
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    <h4 style="text-align: center;">Plasmid Prep</h4>
−
<ul>
+
<ol>
−
<li>Protocols</li>
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<li>Centrifuge @13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorf’s </li>
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<li>Experiments</li>
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<li>Discard supernatant and add 1.9 mL of culture and centrifuge again @13,200 rpm for 10 min </li>
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<li>Documentation of the development of our project</li>
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<li>Re-suspend pelleted cells in P1 Buffer (250µl)  </li>
−
</ul>
+
<li>Add 250µL of P2 buffer & invert 4-6 times (turns homogenous blue) </li>
 +
<li>Add 350µL of N3 & mix by inverting (turns colourless) </li>
 +
<li>Centrifuge @ 10min 13,000 rpm to obtain a pellet </li>
 +
<li>Transfer supernatant in QIA Prep Spin Column by pipetting </li>
 +
<li>Centrifuge 30-60 sec - discard flow through </li>
 +
<li>Wash QIA Prep Spin Column with 0.5mL of PB </li>
 +
<li>Centrifuge for 30-60 seconds - discard flow through </li>
 +
<li>Wash spin column by adding 0.75 mL PE buffer </li>
 +
<li>Centrifuge for 30-60 seconds - discard flow through and centrifuge for a further 1min </li>
 +
<li>Place QIAPrep Column in clean Eppendorf </li>
 +
<li>Elute DNA by adding 30µl of water, stand for 1min, centrifuge for 1min </li>
 +
<li>Plasmid concentration is measured using NanoDrop 2000 spectrophotometer </li>
 +
</ol><br></br>
  
−
<h4>Inspiration</h4>
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    <h4 style="text-align: center;">General PCR Recipe</h4>
−
<ul>
+
<ul>
−
<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia</a></li>
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<li>4 µL of 5x phusion buffer. </li>
−
<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial</a></li>
+
<li>0.4 µL dNTPs. </li>
−
<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech</a></li>
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<li>0.6 µL of DMSO. </li>
−
</ul>
+
<li>0.2 µL of polymerase. </li>
 +
<li>11.8 µL of water. </li>
 +
</ul>
 +
<b>To this mixture, add:</b>
 +
<ul>
 +
<li>1 µL of forward primer. </li>
 +
<li>1 µL of reverse primer. </li>
 +
<li>1 µL of template. </li>
 +
<li>Total volume = 20 µL </li>
 +
</ul><br><br><br>
  
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    <h4 style="text-align: center;">PCR settings: A general program for PCR is as follows:</h4>
 +
<ul>
 +
<li>Initial denaturation at 98<sup>o</sup>C for 30seconds.</li>
 +
</ul>
 +
<b>Followed by 30 repeats of:</b>
 +
<ul>
 +
<li>Denaturation – 98<sup>o</sup>C for 10seconds. </li>
 +
<li>Annealing – 60<sup>o</sup>C for 10seconds. </li>
 +
<li>Extension – 72<sup>o</sup>C for 2minutes. </li>
 +
<li>Final extension – 72<sup>o</sup>C for 10minutes. </li>
 +
</ul><br></br>
  
 +
    <h4 style="text-align: center;">Transformation By Heat Shock</h4>
 +
<ol>
 +
<li>Obtain competent cells from -80<sup>o</sup>C. </li>
 +
<li>Defrost gently on ice. 100µl is sufficient for 2 transformations. </li>
 +
<li>Add 1-10µl of plasmid DNA/ ligation mix to each tube. Incubate on ice for 30 min. </li>
 +
<li>Heat shock in 42<sup>o</sup>C water bath for 30 seconds, then back on ice for 2 min. </li>
 +
<li>Add 200µl of SOC media to each tube, and incubate in the 37<sup>o</sup>C shaker for 1h </li>
 +
<li>For each tube of cells, spread 50µl onto one LB plate with appropriate antibiotic, and 100µl onto a second plate, using aseptic technique. Place your plate upside-down in the 37<sup>o</sup>C incubator. </li>
 +
</ol>
 +
<h6>LB Agar Plate Preparation</h6>
 +
<b>Ingredients:</b> 15g Bacto agar, 1000mL LB media <br>
 +
<b>Methods:</b> Dissolve agar in LB media, autoclave for 15 mins at 121<sup>o</sup>C; wait until cool to touch, then add appropriate antibiotics before pouring into petri dishes.
 +
<h6>LB Media</h6>
 +
<b>Ingredients:</b> Tryptone 10g, Yeast extract 5g, NaCl 10g, Milli-Q water to 1000mL.<br>
 +
<b>Methods:</b> Dissolved 10g tryptone, 5g yeast extract and 10g NaCl in 800mL Milli-Q water, making use of a magnetic stirrer. Once dissolved, bring volume up to 1 L using Milli-Q water. Autoclave 1L of the solution (121<sup>o</sup>C, 15 min, standard liquid cycle).
 +
<h6>SOC Media </h6>
 +
<b>Ingredients:</b> 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl 5M NaCl, 417µL 3M KCl, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose & 500mL Milli-Q water. <br>
 +
<b>Methods:</b> The adjusted quantities were combined in a 1 L measuring column with constant stirring and then placed in the autoclave for sterilisation (121<sup>o</sup>C, 15 min, standard liquid cycle).<br></br>
  
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    <h4 style="text-align: center;">Protein Expression</h4>
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<ol>
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xmlns:w="urn:schemas-microsoft-com:office:word"
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<li>Re-transform restriction-digest screened plasmids according to the heat shock transformation protocol. </li>
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<li>Screen plates for transformant colonies and place in 5mL of ZYM-5052 broth with appropriate antibiotic concentration at 37<sup>o</sup>C with shaking until OD600¬ is 2.0 – 3.0. </li>
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<li>Take whole volume and place in 45mL of ZYM-5052 broth<sup>(1)</sup> in a sterile conical flask containing appropriate antibiotic concentration. Incubate at 37<sup>o</sup>C with shaking. </li>
 +
<li>Cell pellets were collected through centrifugation at 12000 rpm for 5 mins.  </li>
 +
<li>Cell pellets were resuspended in re-suspension buffer (Containing: 50mM Tricine NaOH pH 8.0, 2mM MgCl2, 1mM DTT) and whole lysate was collected through a French Press for functional assays of operon protein products in lysate. </li>
 +
</ol>
 +
<h6>Auto induction Medium (ZYM-5052) </h6>
 +
<ul>
 +
<li>ZY media 9.57ml  </li>
 +
<li>1m MgSO4 20ul </li>
 +
<li>50x5052 200ul </li>
 +
<li>50 x M 200ul </li>
 +
</ul>
 +
<u>1. 50 x M</u>
 +
<ul>
 +
<li>DI water 80ml </li>
 +
<li>Na2HPO4 17.75g </li>
 +
<li>KH2PO4 17g </li>
 +
<li>NH4Cl 13.4 </li>
 +
<li>Na2SO4 3.55g </li>
 +
</ul>
 +
<u>2. 50 x 5052</u>
 +
<ul>
 +
<li>Glycerol 25g (weigh in Beaker) </li>
 +
<li>DI water 73ml </li>
 +
<li>Glucose 2.5g </li>
 +
<li>alpha lactose 10g </li>
 +
</ul>
 +
<u>3. 1M MgSO4</u>
 +
<ul>
 +
<li>MgSO4.7H2O 24.65g </li>
 +
<li>DI water 87ml  </li>
 +
</ul>
 +
<u>4. ZY Media (400mL)</u>
 +
<ul>
 +
<li>Trypton 4g </li>
 +
<li>Yeast extract 2g </li>
 +
</ul>
 +
<br></br>
  
−
<head>
+
    <h4 style="text-align: center;">MgPE Enzymatic Functional Assay</h4>
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<meta name=Title content="">
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<ol>
−
<meta name=Keywords content="">
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<li>Collect whole lysate, from induced operon 3 containing ChlM expressed proteins, using ChlM pET<sup>(2)</sup> as a negative control. </li>  
−
<meta http-equiv=Content-Type content="text/html; charset=macintosh">
+
<li>Prepare a master mix of recombinant ChlM, 25µl of S-Adenosyl-L- methionine and 65µl of 2µM of Mg-protoporphyrin IX. </li>
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<li>Incubate at 30℃ with gentle agitation for 8-10min </li>
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<li>100µL of acetone was added to precipitate protein. </li>  
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<li>The sample was spun down, to separate the protein and supernatant.
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<link rel=File-List href="protocol_files/filelist.xml">
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<li>Supernatant was then subjected to UHPLC to and observed peaks.  </li>
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{font-family:"Noteworthy Light";
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panose-1:2 0 4 0 0 0 0 0 0 0;
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mso-font-charset:0;
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−
mso-generic-font-family:auto;
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−
mso-font-pitch:variable;
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−
mso-font-signature:3 0 0 0 1 0;}
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−
/* Style Definitions */
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−
p.MsoNormal, li.MsoNormal, div.MsoNormal
+
−
{mso-style-name:"Normal\,normal";
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−
mso-style-parent:"";
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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line-height:115%;
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mso-pagination:widow-orphan;
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font-size:11.0pt;
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font-family:Arial;
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−
mso-fareast-font-family:Arial;
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−
mso-bidi-font-family:Arial;
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color:black;}
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h1
+
−
{mso-style-link:"Heading 1 Char";
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−
mso-style-next:"Normal\,normal";
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:1;
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font-size:16.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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−
color:black;
+
−
mso-font-kerning:0pt;
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−
font-weight:normal;}
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h1.CxSpFirst
+
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{mso-style-link:"Heading 1 Char";
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mso-style-next:"Normal\,normal";
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−
mso-style-type:export-only;
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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line-height:115%;
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−
mso-pagination:widow-orphan lines-together;
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−
page-break-after:avoid;
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−
mso-outline-level:1;
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font-size:16.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:black;
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mso-font-kerning:0pt;
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−
font-weight:normal;}
+
−
h1.CxSpMiddle
+
−
{mso-style-link:"Heading 1 Char";
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−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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−
line-height:115%;
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−
mso-pagination:widow-orphan lines-together;
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−
page-break-after:avoid;
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−
mso-outline-level:1;
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font-size:16.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:black;
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mso-font-kerning:0pt;
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font-weight:normal;}
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h1.CxSpLast
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−
{mso-style-link:"Heading 1 Char";
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−
mso-style-next:"Normal\,normal";
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−
mso-style-type:export-only;
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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−
line-height:115%;
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mso-pagination:widow-orphan lines-together;
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−
page-break-after:avoid;
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−
mso-outline-level:1;
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font-size:16.0pt;
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−
font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:black;
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−
mso-font-kerning:0pt;
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−
font-weight:normal;}
+
−
h2
+
−
{mso-style-link:"Heading 2 Char";
+
−
mso-style-next:"Normal\,normal";
+
−
margin:0cm;
+
−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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−
line-height:115%;
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−
mso-pagination:widow-orphan lines-together;
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−
page-break-after:avoid;
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−
mso-outline-level:2;
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font-size:13.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
+
−
color:black;}
+
−
h2.CxSpFirst
+
−
{mso-style-link:"Heading 2 Char";
+
−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
+
−
margin:0cm;
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−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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line-height:115%;
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−
mso-pagination:widow-orphan lines-together;
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−
page-break-after:avoid;
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−
mso-outline-level:2;
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−
font-size:13.0pt;
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−
font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
+
−
mso-bidi-font-family:"Trebuchet MS";
+
−
color:black;}
+
−
h2.CxSpMiddle
+
−
{mso-style-link:"Heading 2 Char";
+
−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
+
−
margin:0cm;
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−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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−
line-height:115%;
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−
mso-pagination:widow-orphan lines-together;
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−
page-break-after:avoid;
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mso-outline-level:2;
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font-size:13.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:black;}
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−
h2.CxSpLast
+
−
{mso-style-link:"Heading 2 Char";
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−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:2;
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font-size:13.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:black;}
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h3
+
−
{mso-style-link:"Heading 3 Char";
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−
mso-style-next:"Normal\,normal";
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−
margin:0cm;
+
−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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−
line-height:115%;
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mso-pagination:widow-orphan lines-together;
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−
page-break-after:avoid;
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−
mso-outline-level:3;
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font-size:12.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;}
+
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h3.CxSpFirst
+
−
{mso-style-link:"Heading 3 Char";
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mso-style-next:"Normal\,normal";
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−
mso-style-type:export-only;
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:3;
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font-size:12.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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−
mso-bidi-font-family:"Trebuchet MS";
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color:#666666;}
+
−
h3.CxSpMiddle
+
−
{mso-style-link:"Heading 3 Char";
+
−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
+
−
margin:0cm;
+
−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:3;
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font-size:12.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;}
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h3.CxSpLast
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{mso-style-link:"Heading 3 Char";
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mso-style-next:"Normal\,normal";
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mso-style-type:export-only;
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margin:0cm;
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margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:3;
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font-size:12.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;}
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h4
+
−
{mso-style-link:"Heading 4 Char";
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−
mso-style-next:"Normal\,normal";
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:4;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;
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text-decoration:underline;
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text-underline:single;}
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h4.CxSpFirst
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{mso-style-link:"Heading 4 Char";
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mso-style-next:"Normal\,normal";
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−
mso-style-type:export-only;
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margin:0cm;
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margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:4;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;
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text-decoration:underline;
+
−
text-underline:single;}
+
−
h4.CxSpMiddle
+
−
{mso-style-link:"Heading 4 Char";
+
−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
+
−
margin:0cm;
+
−
margin-bottom:.0001pt;
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mso-add-space:auto;
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−
line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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−
mso-outline-level:4;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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−
font-weight:normal;
+
−
text-decoration:underline;
+
−
text-underline:single;}
+
−
h4.CxSpLast
+
−
{mso-style-link:"Heading 4 Char";
+
−
mso-style-next:"Normal\,normal";
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−
mso-style-type:export-only;
+
−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:4;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;
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text-decoration:underline;
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text-underline:single;}
+
−
h5
+
−
{mso-style-link:"Heading 5 Char";
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−
mso-style-next:"Normal\,normal";
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−
margin:0cm;
+
−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:5;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;}
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−
h5.CxSpFirst
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−
{mso-style-link:"Heading 5 Char";
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mso-style-next:"Normal\,normal";
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−
mso-style-type:export-only;
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margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:5;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;}
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h5.CxSpMiddle
+
−
{mso-style-link:"Heading 5 Char";
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−
mso-style-next:"Normal\,normal";
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−
mso-style-type:export-only;
+
−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:5;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;}
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−
h5.CxSpLast
+
−
{mso-style-link:"Heading 5 Char";
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−
mso-style-next:"Normal\,normal";
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−
mso-style-type:export-only;
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:5;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;}
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h6
+
−
{mso-style-link:"Heading 6 Char";
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−
mso-style-next:"Normal\,normal";
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−
margin:0cm;
+
−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:6;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;
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font-style:italic;}
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h6.CxSpFirst
+
−
{mso-style-link:"Heading 6 Char";
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−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
+
−
margin:0cm;
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−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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−
page-break-after:avoid;
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mso-outline-level:6;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;
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−
font-style:italic;}
+
−
h6.CxSpMiddle
+
−
{mso-style-link:"Heading 6 Char";
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−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
+
−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:6;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;
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−
font-style:italic;}
+
−
h6.CxSpLast
+
−
{mso-style-link:"Heading 6 Char";
+
−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
+
−
margin:0cm;
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−
margin-bottom:.0001pt;
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mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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mso-outline-level:6;
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font-size:11.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:#666666;
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font-weight:normal;
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font-style:italic;}
+
−
p.MsoTitle, li.MsoTitle, div.MsoTitle
+
−
{mso-style-link:"Title Char";
+
−
mso-style-next:"Normal\,normal";
+
−
margin:0cm;
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−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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page-break-after:avoid;
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font-size:21.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:black;}
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−
p.MsoTitleCxSpFirst, li.MsoTitleCxSpFirst, div.MsoTitleCxSpFirst
+
−
{mso-style-link:"Title Char";
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−
mso-style-next:"Normal\,normal";
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−
mso-style-type:export-only;
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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line-height:115%;
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mso-pagination:widow-orphan lines-together;
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−
page-break-after:avoid;
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font-size:21.0pt;
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font-family:"Trebuchet MS";
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mso-fareast-font-family:"Trebuchet MS";
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mso-bidi-font-family:"Trebuchet MS";
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color:black;}
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−
p.MsoTitleCxSpMiddle, li.MsoTitleCxSpMiddle, div.MsoTitleCxSpMiddle
+
−
{mso-style-link:"Title Char";
+
−
mso-style-next:"Normal\,normal";
+
−
mso-style-type:export-only;
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−
margin:0cm;
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−
margin-bottom:.0001pt;
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−
mso-add-space:auto;
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−
{mso-level-number-format:alpha-lower;
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−
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−
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−
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−
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−
{mso-level-number-format:roman-lower;
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−
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−
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−
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−
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−
{mso-level-tab-stop:none;
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−
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−
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−
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−
{mso-level-number-format:alpha-lower;
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−
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−
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−
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−
{mso-level-number-format:roman-lower;
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−
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−
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−
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−
{mso-level-tab-stop:none;
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−
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−
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−
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−
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−
{mso-level-number-format:alpha-lower;
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−
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−
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−
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−
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−
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−
{mso-level-number-format:roman-lower;
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−
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−
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−
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−
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−
mso-list-template-ids:1017046104;}
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−
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−
{mso-level-tab-stop:none;
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−
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−
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−
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−
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−
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−
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−
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−
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−
{mso-level-number-format:alpha-lower;
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−
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−
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−
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−
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−
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−
@list l3:level3
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−
{mso-level-number-format:roman-lower;
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−
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−
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−
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−
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−
@list l3:level4
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−
{mso-level-tab-stop:none;
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−
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−
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−
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−
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−
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−
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−
@list l3:level5
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−
{mso-level-number-format:alpha-lower;
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−
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−
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−
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−
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−
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−
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−
@list l3:level6
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−
{mso-level-number-format:roman-lower;
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−
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−
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−
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−
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−
@list l3:level7
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−
{mso-level-tab-stop:none;
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−
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−
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−
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−
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−
@list l3:level8
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−
{mso-level-number-format:alpha-lower;
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−
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−
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−
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−
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−
{mso-level-number-format:roman-lower;
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
{mso-level-number-format:bullet;
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
{mso-level-number-format:bullet;
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
@list l6:level9
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
mso-level-text:\25CF;
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−
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−
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−
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−
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−
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−
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text-indent:-18.0pt;}
+
−
@list l16:level8
+
−
{mso-level-tab-stop:288.0pt;
+
−
mso-level-number-position:left;
+
−
text-indent:-18.0pt;}
+
−
@list l16:level9
+
−
{mso-level-tab-stop:324.0pt;
+
−
mso-level-number-position:left;
+
−
text-indent:-18.0pt;}
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−
@list l17
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−
{mso-list-id:1390767940;
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−
mso-list-template-ids:-916832514;}
+
−
@list l17:level1
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−
{mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:18.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l17:level2
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−
{mso-level-number-format:alpha-lower;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:54.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l17:level3
+
−
{mso-level-number-format:roman-lower;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:right;
+
−
text-indent:90.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l17:level4
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−
{mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:126.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l17:level5
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−
{mso-level-number-format:alpha-lower;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:162.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l17:level6
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−
{mso-level-number-format:roman-lower;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:right;
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−
text-indent:198.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l17:level7
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−
{mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:234.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l17:level8
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−
{mso-level-number-format:alpha-lower;
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−
mso-level-tab-stop:none;
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−
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−
text-indent:270.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l17:level9
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−
{mso-level-number-format:roman-lower;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:right;
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−
text-indent:306.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l18
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−
{mso-list-id:1391926166;
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−
mso-list-template-ids:305986318;}
+
−
@list l18:level1
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−
{mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:18.0pt;
+
−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l18:level2
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−
{mso-level-number-format:alpha-lower;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
+
−
text-indent:54.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
+
−
@list l18:level3
+
−
{mso-level-number-format:roman-lower;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:right;
+
−
text-indent:90.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l18:level4
+
−
{mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:126.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l18:level5
+
−
{mso-level-number-format:alpha-lower;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
+
−
text-indent:162.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
+
−
@list l18:level6
+
−
{mso-level-number-format:roman-lower;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:right;
+
−
text-indent:198.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l18:level7
+
−
{mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:234.0pt;
+
−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l18:level8
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−
{mso-level-number-format:alpha-lower;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:270.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l18:level9
+
−
{mso-level-number-format:roman-lower;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:right;
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−
text-indent:306.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l19
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−
{mso-list-id:1442073371;
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−
mso-list-template-ids:410680830;}
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−
@list l19:level1
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CF;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
+
−
text-indent:18.0pt;
+
−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l19:level2
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−
{mso-level-number-format:bullet;
+
−
mso-level-text:\25CB;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:54.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l19:level3
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25A0;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:90.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l19:level4
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CF;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:126.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l19:level5
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CB;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:162.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l19:level6
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25A0;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:198.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l19:level7
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CF;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:234.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l19:level8
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CB;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:270.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l19:level9
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25A0;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:306.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l20
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−
{mso-list-id:1572617164;
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−
mso-list-template-ids:-1956855408;}
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−
@list l20:level1
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CF;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
+
−
text-indent:18.0pt;
+
−
mso-ansi-font-size:12.0pt;
+
−
mso-ascii-font-family:Arial;
+
−
mso-fareast-font-family:Arial;
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−
mso-hansi-font-family:Arial;
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−
mso-bidi-font-family:Cambria;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l20:level2
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CB;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:54.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l20:level3
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25A0;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:90.0pt;
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−
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l20:level4
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CF;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:126.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l20:level5
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CB;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:162.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l20:level6
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25A0;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:198.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l20:level7
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CF;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:234.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
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−
@list l20:level8
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25CB;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:270.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
+
−
@list l20:level9
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−
{mso-level-number-format:bullet;
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−
mso-level-text:\25A0;
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−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
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−
text-indent:306.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
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−
text-line-through:none;}
+
−
@list l21
+
−
{mso-list-id:1580409131;
+
−
mso-list-template-ids:1448512234;}
+
−
@list l21:level1
+
−
{mso-level-tab-stop:none;
+
−
mso-level-number-position:left;
+
−
text-indent:18.0pt;
+
−
mso-text-animation:none;
+
−
text-decoration:none;
+
−
text-underline:none;
+
−
text-decoration:none;
+
−
text-line-through:none;}
+
−
@list l21:level2
+
−
{mso-level-number-format:alpha-lower;
+
−
mso-level-tab-stop:none;
+
−
mso-level-number-position:left;
+
−
text-indent:54.0pt;
+
−
mso-text-animation:none;
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−
text-decoration:none;
+
−
text-underline:none;
+
−
text-decoration:none;
+
−
text-line-through:none;}
+
−
@list l21:level3
+
−
{mso-level-number-format:roman-lower;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:right;
+
−
text-indent:90.0pt;
+
−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
+
−
text-line-through:none;}
+
−
@list l21:level4
+
−
{mso-level-tab-stop:none;
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−
mso-level-number-position:left;
+
−
text-indent:126.0pt;
+
−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
+
−
text-line-through:none;}
+
−
@list l21:level5
+
−
{mso-level-number-format:alpha-lower;
+
−
mso-level-tab-stop:none;
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−
mso-level-number-position:left;
+
−
text-indent:162.0pt;
+
−
mso-text-animation:none;
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−
text-decoration:none;
+
−
text-underline:none;
+
−
text-decoration:none;
+
−
text-line-through:none;}
+
−
@list l21:level6
+
−
{mso-level-number-format:roman-lower;
+
−
mso-level-tab-stop:none;
+
−
mso-level-number-position:right;
+
−
text-indent:198.0pt;
+
−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
+
−
text-line-through:none;}
+
−
@list l21:level7
+
−
{mso-level-tab-stop:none;
+
−
mso-level-number-position:left;
+
−
text-indent:234.0pt;
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−
mso-text-animation:none;
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−
text-decoration:none;
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−
text-underline:none;
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−
text-decoration:none;
+
−
text-line-through:none;}
+
−
@list l21:level8
+
−
{mso-level-number-format:alpha-lower;
+
−
mso-level-tab-stop:none;
+
−
mso-level-number-position:left;
+
−
text-indent:270.0pt;
+
−
mso-text-animation:none;
+
−
text-decoration:none;
+
−
text-underline:none;
+
−
text-decoration:none;
+
−
text-line-through:none;}
+
−
@list l21:level9
+
−
{mso-level-number-format:roman-lower;
+
−
mso-level-tab-stop:none;
+
−
mso-level-number-position:right;
+
−
text-indent:306.0pt;
+
−
mso-text-animation:none;
+
−
text-decoration:none;
+
−
text-underline:none;
+
−
text-decoration:none;
+
−
text-line-through:none;}
+
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@list l22
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−
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−
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−
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−
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−
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@list l28
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−
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mso-list-template-ids:625272808;}
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@list l28:level1
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−
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−
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−
{mso-level-number-format:alpha-lower;
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−
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−
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−
{mso-level-number-format:roman-lower;
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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@list l31
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−
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−
mso-list-template-ids:-1074340452;}
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
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−
@list l31:level3
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−
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−
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−
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−
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−
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−
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−
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−
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−
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@list l31:level4
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−
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−
<body bgcolor=white lang=EN-AU style='tab-interval:36.0pt'>
+
 +
    <h4 style="text-align: center;">UHPLC </h4>
 +
<p>The UHPLC was performed according to Sawicki and Willows<sup>(3)</sup>. Briefly, a Shimadzu HPLC system was used at 2ml/min with an Alltech C8 column. A 5 min linear gradient was used for separation of Mg-protoporphyrin IX and Magnesium-protoporphyrin IX mono-methyl ester. Compounds were detected at an excitation wavelength of excitation of 416nm and emission of 595nm.  </p>
 +
<br></br>  
  
−
<div class=Section1>
+
    <h4 style="text-align: center;">SDS-PAGE Electrophoresis</h4>
 +
<ol>
 +
<li>Re-suspend pelleted bacterial cells in 200µL of Milli-Q-water </li>
 +
<li>Transfer 50 µL of suspension into new Eppendorf tubes and combine with 50ul of 2xTruSep sample buffer. </li>
 +
<li> Shear the cells using a Hamilton syringe. </li>
 +
<li>Centrifuge the preparation for 3minutes @ 13,000 rpm. </li>
 +
<li>Load 20 µL of the supernatant into gel. </li>
 +
<li>Conduct electrophoresis at a constant voltage (200V) for 1 hour. </li>
 +
<li>Coommassie Stain for ~30 minutes. </li>
 +
</ol><br></br>
  
−
<p class=MsoNormal><u>Protocols </u></p>
 
  
−
<p class=MsoNormal align=center style='text-align:center'><u><span
+
    <h4 style="text-align: center;"> Gun4 PPIX binding by fluorescence spectrophotometry </h4>
−
style='font-size:14.0pt;line-height:115%'>Making competent cells</span></u></p>
+
<h6>Preparation of PPIX</h6>
 +
<ol>
 +
<li>Approximately 1mg of dried PPIX was dissolved in 5ul of 1M NaOH, and diluted with 200ul of deionized water.</li>
 +
<li>Pellet any undissolved particulate by centrifugation, and dilute the supernatant 100-fold with 5% HCl.</li>
 +
<li>The concentration of the PPIX solution was calculated by measuring the absorbance of the diluted solution at 404nm, using the Beer-Lambert equation with molar extinction coefficient of 278000.</li>
 +
<li>A 2uM concentration solution of PPIX was used for the binding assay.</li>
 +
</ol>
  
−
<p class=MsoNormal><o:p>&nbsp;</o:p></p>
+
<h6>Gun4 PPIX binding </h6>
 +
<ol>
 +
<li>Over-expressed Gun4 containing cell lysates was prepared as described above.</li>  
 +
<li>The protein concentration was estimated using Bradford reagent, and the lysate diluted to approximately 0.2mg/ml.</li>
 +
<li>100ul of the 2uM PPIX solution was added with varying amounts of protein lysate to a total volume of 200ul, and transferred to a clean quartz cuvette for fluorescence measurements in a Cory Eclipse fluorescence spectrophotometer (Agilent)</li>
 +
<li> The excitation wavelength was set at 280nm and the emission at measure from 580nm to 800nm. The emission maxima for protein bound PPIX is at 635nm.</li>
 +
</ol>
 +
<br><br>
 +
<h5>References</h5>
 +
<ol>
  
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:49.65pt;mso-add-space:
+
<li>Studier, F. W. (2014). Stable expression clones and auto-induction for protein production in E. coli. In <i>Structural Genomics</i> (pp. 17-32). Humana Press.</li>
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Using a sterile
+
−
plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate.
+
−
Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22<sup>o</sup>C,
+
−
200-250rpm.<o:p></o:p></span></p>
+
  
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:14.2pt;mso-add-space:
+
<li>Meinecke, L., Alawady, A., Schroda, M., Willows, R., Kobayashi, M. C., Niyogi, K. K., ... & Beck, C. F. (2010). Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX. <i>Plant molecular biology, 72</i>(6), 643-658.</li>
−
auto;text-align:justify;text-indent:-.55pt'><span style='font-size:14.0pt'><o:p>&nbsp;</o:p></span></p>
+
  
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:49.65pt;mso-add-space:
+
<li>Sawicki, A., & Willows, R. (2007). S-Adenosyl-L-methionine: magnesium-protoporphyrin IX O-methyltransferase from Rhodobacter capsulatus: mechanistic insights and stimulation with phospholipids. <i>Biochem. J, 406</i>, 469-478. </li>
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>A600 should be
+
−
0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~
+
−
0.5.<o:p></o:p></span></p>
+
  
−
<p class=normalcxspmiddleCxSpLast style='margin-left:14.2pt;mso-add-space:auto;
 
−
text-align:justify;text-indent:-.55pt'><span style='font-size:14.0pt'><o:p>&nbsp;</o:p></span></p>
 
  
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
</ol>
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
<br>
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>3.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Remove the flask from the incubator and place on ice
+
−
for 10 minutes.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>4.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Transfer the culture to a 15mL centrifuge tube and
+
−
spin at 2500 x g for 10 min at 4<sup>o</sup>C<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>5.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Pour off and discard the supernatant, and immediately
+
−
place the tube on ice.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>6.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Resuspend your cells in 1mL of ice-cold TB buffer,
+
−
make sure there are no clumps of cells left, but also treat your cells gently
+
−
and keep them cold.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>7.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Add ice-cold TB buffer to bring volume up to 1/5th of
+
−
the original culture volume (~30mL in this case). Mix the tube by gently
+
−
inverting 3 times.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>8.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Incubate the tube on ice for 10 minutes.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>9.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Centrifuge at 2<span class=GramE>,500 x</span> g for 7
+
−
minutes at 4<sup>o</sup>C, discard the supernatant.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>10.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Gently resuspend
+
−
the cells in ~1/20th of the original culture volume of ice-cold TB buffer.
+
−
NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. <span
+
−
class=GramE>e</span>.g. if the culture OD600 was 0.1 then resuspend in 1/100th
+
−
of original volume.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>11.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Pre-chill 1.5ml <span
+
−
class=SpellE>Eppendorf</span> tubes on ice. Add 930µl of your cell suspension,
+
−
keeping the remainder on ice in the 15mL tube.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>12.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Add 70µl of DMSO
+
−
to the 930µl of cell suspension. Mix gently by swirling, and place on ice.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>13.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Aliquot 100µl of
+
−
the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the
+
−
tubes with: Date &#8211; Strain. <span class=GramE>Snap freeze</span> with
+
−
liquid nitrogen.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='margin-left:49.65pt;text-align:justify;text-indent:
+
−
-18.0pt;line-height:normal;mso-list:l16 level1 lfo1'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>14.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Repeat step 11-13
+
−
for the rest of your cell suspension in step 10. Store cells at -80<sup>o</sup>C<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.97a2w31llx4p></a><o:p><span style='text-decoration:none'>&nbsp;</span></o:p></h4>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.33k2zqtujk1j></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333'>TB BUFFER</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l7 level1 lfo2'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span
+
−
style='font-size:14.0pt'> 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g <span
+
−
class=SpellE>KCl</span>.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l7 level1 lfo2'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span
+
−
style='font-size:14.0pt'> All components (except for MnCl2-4H2O) were mixed and
+
−
dissolved in 500 <span class=SpellE>mL</span> of water and pH adjusted to 6.7
+
−
with KOH. Then, MnCl2-4H2O, was dissolved in 300 <span class=SpellE>mL</span>
+
−
of water, mixed and solution adjusted to 1 L. Sterilisation via filtration
+
−
followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.77uif8qp8qn4></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333'>EDTA BUFFER</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l0 level1 lfo3'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span
+
−
style='font-size:14.0pt'> 37.22g EDTA solid, 180 <span class=SpellE>mL</span>
+
−
of water and pH adjusted to 8.0 using 10M <span class=SpellE>NaOH</span>.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l0 level1 lfo3'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span
+
−
style='font-size:14.0pt'> Components were combined then pH adjusted.<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.q84g5o6q56lo></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333'>TAE BUFFER</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l23 level1 lfo4'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span
+
−
style='font-size:14.0pt'> 121.2g <span class=SpellE>Tris</span> base (dissolved
+
−
in water) with 28.55mL of glacial acetic acid &amp; 50mL 0.5M EDTA (pH 8.0).<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l23 level1 lfo4'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span
+
−
style='font-size:14.0pt'> A total volume of 500 <span class=SpellE>mL</span>
+
−
was made up as a 50x stock solution using all components<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.2oiikqt7my41></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333'>Making agarose gel</span></b></h4>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.ffz7g0pkhkr5></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333'>Preparing the Gel </span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l17 level1 lfo5'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Mix 1g of agarose powder with 100ml of 1x TAE buffer
+
−
and heat for 1minute or until all agarose is dissolved.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l17 level1 lfo5'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Wait until it has
+
−
cooled (not set), and add 1ul of <span class=SpellE>GelRed</span> into the
+
−
mixture. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l17 level1 lfo5'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Pour the solution
+
−
into a cast with an appropriate comb.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l17 level1 lfo5'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Leave to set.<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.s2q8apkb2g></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333'>Running the Gel </span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l18 level1 lfo6'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Mix 1ul of 1kbp DNA ladder with 6ul of loading dye (<span
+
−
class=SpellE>bromophenol</span> blue) and 4ul of 1x TAE buffer (total 6ul) and
+
−
load onto first well <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l18 level1 lfo6'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Mix 5ul of PCR
+
−
products with 1ul of loading dye and load onto wells.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l18 level1 lfo6'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Run gel at 90V
+
−
for 45minutes approximately<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l18 level1 lfo6'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Photograph gels
+
−
under UV light<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
style='font-size:14.0pt;line-height:160%'>Composite part ligation<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
style='font-size:14.0pt;line-height:160%'>The assembly of composite parts from
+
−
two existing <span class=SpellE>BioBricks</span> i.e. <span class=SpellE>BioBrick</span>
+
−
plasmid A (parent vector) and <span class=SpellE>BioBrick</span> plasmid B
+
−
(gene to be inserted) was performed through a restriction digest/ligation
+
−
protocol. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l3 level1 lfo7'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>200ng of each <span
+
−
class=SpellE>BioBrick</span> plasmid was digested as follows: Plasmid <span
+
−
class=SpellE>BioBrick</span> A with NEB restriction enzymes <span class=SpellE>SpeI</span>
+
−
and <span class=SpellE>PstI</span><span class=GramE>;</span> plasmid <span
+
−
class=SpellE>BioBrick</span> B with <span class=SpellE>XbaI</span> and <span
+
−
class=SpellE>PstI</span>, according to supplier protocol. (1hr @37<sup>o</sup>C,
+
−
then 20mins @80<sup>o</sup>C).<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l3 level1 lfo7'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>1µL of <span
+
−
class=GramE>Fast</span> alkaline <span class=SpellE>phosphatase</span> (Thermo
+
−
Scientific) was added to reaction tube of plasmid <span class=SpellE>BioBrick</span>
+
−
A to <span class=SpellE>dephosphorylate</span> the gene fragments and prevent
+
−
re-ligation of the parent vector. Reaction tubes were incubated at 37<sup>o</sup>C
+
−
for 60 mins, followed by a deactivation step at 80<sup>o</sup>C for 20 mins.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l3 level1 lfo7'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Ligation was then
+
−
performed with an insert to vector molar ratio of 3:1. 1µL of T4 ligase (NEB)
+
−
was added to the mix for ligation, according to supplier protocol. Ligation
+
−
reactions were performed at 37<sup>o</sup>C for 60 mins and kept on ice for
+
−
transformation into chemical competent cells.<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.j91d528avia></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>MANUAL TRYPSIN IN-GEL DIGESTION PROTOCOL FOR COOMASSIE STAINED GELS</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
class=SpellE><span style='font-size:14.0pt'>Coommassie</span></span><span
+
−
style='font-size:14.0pt'> de-stain gels: by washing briefly with 200µL NH4HCO3
+
−
(Solution A) to make sure gel pieces are at the correct pH. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Add 200µL 50% <span
+
−
class=SpellE>Acetonitrile</span> / 50% 100mM NH4HCO3 (Solution B) into each
+
−
well. Vortex to mix and incubate for 10 minutes. Remove liquid.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Repeat step 2.
+
−
Gel pieces should be clear at this stage. If they are still blue, repeat as
+
−
necessary until <span class=SpellE>color</span> is gone.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Wash for 5 min
+
−
with 50µL of 100% <span class=SpellE>Acetonitrile</span> (Solution C) to
+
−
dehydrate gel pieces. Vortex during incubation.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Remove <span
+
−
class=SpellE>Acetonitrile</span>, <span class=GramE>then</span> let air-dry for
+
−
10 min. The gel pieces should be noticeably shrunken and probably white.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Reduction and
+
−
Alkylation: Cover gel pieces with 50µL 10mM DTT in 50mM NH4HCO3 (Solution D).
+
−
Let proteins reduced for 45-60 min in 37<sup>o</sup>C incubator.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Remove DTT solution
+
−
and add 50µL of 55mM <span class=SpellE>iodoacetamide</span> in 50mM NH4HCO3
+
−
(Solution E). Incubate for 45 min in dark place at room temperature.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>8.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span class=GramE><span style='font-size:14.0pt'>Remove
+
−
<span class=SpellE>iodoacetamide</span></span></span><span style='font-size:
+
−
14.0pt'>, <span class=GramE>discard</span>.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>9.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Wash gel pieces
+
−
with 200ul of NH4HCO3 (Solution A) for 5 min with <span class=SpellE>vortexing</span>.
+
−
<span class=GramE>before</span> adding 100ul of 100% <span class=SpellE>Acetonitrile</span>
+
−
(Solution C).<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>10.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span class=GramE><span style='font-size:14.0pt'>Remove
+
−
liquid after 5 min</span></span><span style='font-size:14.0pt'>, <span
+
−
class=GramE>discard</span>.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>11.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Wash gel pieces
+
−
with 50ul 100mM NH4HCO3 (Solution A) for 10 min, then twice with 200ul 50% <span
+
−
class=SpellE>Acetonitrile</span> / 50% 100mM NH4HCO3 (Solution B) for 10min<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>12.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Dehydrate with
+
−
100 <span class=SpellE>ul</span> 100% <span class=SpellE>Acetonitrile</span>
+
−
(Solution C) for 5 min as above.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>13.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Remove remaining
+
−
liquid and let the gel dried.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>14.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span class=SpellE><span style='font-size:14.0pt'>Trypsin</span></span><span
+
−
style='font-size:14.0pt'> Digestion: Prepare <span class=SpellE>trypsin</span>
+
−
mix to final concentration of 13ng/µL in 50mM Ammonium bicarbonate.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>15.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Add 30µL (or more
+
−
if required) of mixed <span class=SpellE>trypsin</span> solution to cover the
+
−
gel pieces. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>16.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Allow 30 min for
+
−
gel rehydration at 4<sup>o</sup>C (on ice). <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>17.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Digest overnight
+
−
at 37<sup>o</sup>C.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>18.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Peptide
+
−
Extraction: Transfer the digest solution supernatant (if any) into clean 0.65ml
+
−
<span class=SpellE>Eppendorf</span> tubes.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>19.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>To the gel
+
−
pieces, add 50 µL of 50% <span class=SpellE>acetonitrile</span> / 2% formic acid,
+
−
incubate 30 min. Spin, remove supernatant and combine with initial digest
+
−
solution supernatant. Please note that total volume may vary depending on the
+
−
gel sizes.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>20.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Vortex the
+
−
extracted digests, speed vac to reduce volume to 10 µL. If the remaining volume
+
−
is less than 10ul, use 2% formic acid to bring the volume up to 10 µL.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>21.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Spin at 14,000
+
−
rpm for 30 min to remove any micro particulates. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l10 level1 lfo8'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>22.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Transfer the
+
−
supernatant to a fresh 0.65ml <span class=SpellE>eppendorf</span> tube for
+
−
storage at 4<sup>o</sup>C fridge OR directly into PCR plate for Mass spec for
+
−
analysis.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<h4 style='text-align:justify;line-height:160%;mso-pagination:widow-orphan;
+
−
page-break-after:auto'><a name=h.tjzgdytzmhl9></a><b style='mso-bidi-font-weight:
+
−
normal'><span style='font-size:15.0pt;line-height:160%;font-family:Arial;
+
−
mso-fareast-font-family:Arial;mso-bidi-font-family:Arial;color:#333333;
+
−
text-decoration:none;text-underline:none'>Reagents </span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l12 level1 lfo9'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Buffer 100mM <b style='mso-bidi-font-weight:normal'>Ammonium
+
−
Bicarbonate </b><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:118.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l12 level2 lfo9'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9675;<span
+
−
style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>ADD 0.78 g ammonium bicarbonate to 100ml ddH20. Fill
+
−
first medium size reservoir.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l30 level1 lfo10'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Reduction: -<b style='mso-bidi-font-weight:normal'> <span
+
−
class=SpellE>Dithiothreitol</span> (DTT) </b><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l30 level2 lfo10'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9675;<span
+
−
style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Add 15 <span class=SpellE>mL</span> of 100 <span
+
−
class=SpellE>mM</span> ammonium bicarbonate to 23.1 mg of DTT to give a solution
+
−
of 10 <span class=SpellE>mM</span> DTT.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l14 level1 lfo11'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Alkylation <span class=GramE>-<span
+
−
style="mso-spacerun: yes">&nbsp; </span><span class=SpellE><b style='mso-bidi-font-weight:
+
−
normal'>Iodoacetamide</b></span></span><b style='mso-bidi-font-weight:normal'>(DTT)
+
−
</b><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l14 level2 lfo11'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9675;<span
+
−
style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Add 15 <span class=SpellE>mL</span> of 100 <span
+
−
class=SpellE>mM</span> ammonium bicarbonate to 153 mg of <span class=SpellE>iodoacetamide</span>
+
−
to give a solution of 55 <span class=SpellE>mM</span> <span class=SpellE>iodoacetamide</span>
+
−
- 15 <span class=SpellE>mL</span> of each is enough for up to 96 samples.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l5 level1 lfo12'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Peptide Extraction Solution -<b style='mso-bidi-font-weight:
+
−
normal'> 2% Formic acid/ 50% <span class=SpellE>Acetonitrile</span> solution </b><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l5 level2 lfo12'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9675;<span
+
−
style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Add 2000µL of formic acid and 50 <span class=SpellE>mL</span>
+
−
of <span class=SpellE>acetonitrile</span> to 75 <span class=SpellE>mL</span> of
+
−
ddH2O. 15 <span class=SpellE>mL</span> is enough for up to 96 samples.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l24 level1 lfo13'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Dehydration -<b style='mso-bidi-font-weight:normal'> <span
+
−
class=SpellE>Acetonitrile</span> </b><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l24 level2 lfo13'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9675;<span
+
−
style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>A full square reservoir is plenty for up to 96 samples
+
−
with dehydration and/or <span class=SpellE>Coomassie</span> <span class=SpellE>destaining</span>.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l27 level1 lfo14'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
class=SpellE><b style='mso-bidi-font-weight:normal'><span style='font-size:
+
−
14.0pt'>Trypsin</span></b></span><span style='font-size:14.0pt'><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l27 level2 lfo14'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9675;<span
+
−
style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span
+
−
class=SpellE><span style='font-size:14.0pt'>Trypsin</span></span><span
+
−
style='font-size:14.0pt'> is prepared by adding 200µL of <span class=SpellE>Resuspension</span>
+
−
Buffer into one vial containing 20ug of Proteomics grade <span class=SpellE>trypsin</span>.
+
−
<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:118.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l27 level2 lfo14'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9675;<span
+
−
style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Add 1.2ml of 50mM NH4HCO3 into each <span
+
−
class=SpellE>trypsin</span> vial (final concentration of <span class=SpellE>Trypsin</span>
+
−
is 16ng/µL). Mix thoroughly. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:118.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l27 level2 lfo14'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9675;<span
+
−
style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>This will provide enough <span class=SpellE>Trypsin</span>
+
−
for 46 digestions.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt'>Mass Spec<o:p></o:p></span></b></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Samples need to
+
−
be spotted on the 384 spot MALDI plate: Odd rows are used for samples, and even
+
−
rows are for calibration standards. It is important that one sample spot in the
+
−
odd row is coupled by a calibration spot in the even row.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Samples are
+
−
cleaned up with C18 Zip tips before spotting onto the plate. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Start with a
+
−
fresh <span class=SpellE>Ziptip</span>, using a 10 µL pipette, and slowly
+
−
withdraw and dispense to waste 10 µL 90% (v/v) <span class=SpellE>acetonitrile</span>
+
−
/ 0.1 % TFA three times. Do not throw away the tip!<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Slowly withdraw
+
−
and dispense to waste 10 µL 0.1 % TFA five times to equilibrate the C18
+
−
material in aqueous buffer.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Slowly withdraw
+
−
and dispense your first sample (10 µL) ten times. After this step the peptides
+
−
from your digest are bound onto the C18 resin in the tip.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l33 level1 lfo15'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Withdraw and
+
−
discard to waste 10 µL of 0.1 % TFA five times. This washes away unwanted salts
+
−
and contaminants. Make sure you expel all the liquid on the last wash.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><span style="mso-spacerun: yes">&nbsp;&nbsp;&nbsp;
+
−
</span>7. Withdraw 1 µL of matrix solution. This contains 70% <span
+
−
class=SpellE>acetonitrile</span> so it is<span style="mso-spacerun:
+
−
yes">&nbsp;&nbsp;&nbsp; </span>used to elute peptides in the presence of
+
−
matrix. <span class=GramE>(4 mg / <span class=SpellE>mL</span> CHCA
+
−
[&#945;-cyano-4-hydroxycynnamic acid], 70 % <span class=SpellE>MeCN</span>, 0.1
+
−
% TFA).</span> <o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><span style="mso-spacerun:
+
−
yes">&nbsp;&nbsp;&nbsp;&nbsp; </span>8. Spot the eluted peptide sample in
+
−
matrix solution directly onto the MALDI target plate in an odd row, trying not
+
−
to touch the surface of the plate with the tip. Record the location of your
+
−
samples.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><span style="mso-spacerun:
+
−
yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>9. New C18 tip is needed for
+
−
each sample. <o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><span style="mso-spacerun: yes">&nbsp;&nbsp;&nbsp;
+
−
</span>10. BSA digest at 1pmol/µL is used as control. This does not required <span
+
−
class=SpellE>ziptipping</span>.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><span style="mso-spacerun:
+
−
yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>11. Next, prepare calibration
+
−
standards (<span class=SpellE>Pepmix</span> and Matrix solutions at 1:1 ratio),
+
−
and spot 1ul of this onto spots in even row next to your samples. These are
+
−
your near-point calibration standards. <o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><span style="mso-spacerun:
+
−
yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>12.Allow the plate to
+
−
air-dry.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><span style="mso-spacerun:
+
−
yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>13. Plates can be stored in 4<sup>o</sup>C
+
−
fridge and in the dark for up to a week, ready for analysis.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><span style="mso-spacerun:
+
−
yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>14. Samples are <span
+
−
class=SpellE>analyzed</span> using the 4800 Plus MALDI <span class=SpellE>Tof-Tof</span>
+
−
<span class=SpellE>Analyzer</span>.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.eh11zdoajzzn></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>LB AGAR PLATE PREPARATION</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l28 level1 lfo16'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Add 15g <span class=SpellE>Bacto</span> agar to 1000mL
+
−
of LB media and autoclave (121<sup>o</sup>C for 15mins).<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l28 level1 lfo16'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Add 1000uL of <span
+
−
class=SpellE>Chloroamphenicol</span> (25mg/mL), Ampicillin (50mg/mL) or <span
+
−
class=SpellE>Kanamycin</span> (30mg/mL) and mix well before plating out and
+
−
setting the agar.<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.8d6a21bkpjv7></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>LB MEDIA</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l22 level1 lfo17'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span
+
−
style='font-size:14.0pt'> <span class=SpellE>Tryptone</span> 10g, Yeast extract
+
−
5g, <span class=SpellE>NaCl</span> 10g, <span class=SpellE>Milli</span>-Q water
+
−
to 1000mL.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l22 level1 lfo17'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span
+
−
style='font-size:14.0pt'> Dissolved 10g <span class=SpellE>tryptone</span>, 5g
+
−
yeast extract and 10g <span class=SpellE>NaCl</span> in 800mL <span
+
−
class=SpellE>Milli</span>-Q water, making use of a magnetic stirrer. Once
+
−
dissolved, bring volume up to 1 L using <span class=SpellE>Milli</span>-Q
+
−
water. Autoclave 1L of the solution (121<sup>o</sup>C, 15 min, standard liquid
+
−
cycle). <o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.msyjoqpi6c3m></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>SOC MEDIA (FOR COMPETENT CELLS)</span></b></h4>
+
−
 
+
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<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l20 level1 lfo18'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Ingredients:</span></b><span
+
−
style='font-size:14.0pt'> 10g <span class=SpellE>bacto-tryptone</span>, 2.5g <span
+
−
class=SpellE>bacto</span>-yeast, 1000µl 5M <span class=SpellE>NaCl</span>,
+
−
417µL 3M <span class=SpellE>KCl</span>, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose
+
−
&amp; 500mL <span class=SpellE>Milli</span>-Q water. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l20 level1 lfo18'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:14.0pt'>Methods:</span></b><span
+
−
style='font-size:14.0pt'> The adjusted quantities were combined in a 1 L
+
−
measuring column with constant stirring and then placed in the autoclave for
+
−
sterilisation (121<sup>o</sup>C, 15 min, standard liquid cycle).<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.q141kogjduod></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>NANODROP for THERMO SCIENTIFIC <span class=SpellE>NanoDrop</span> 2000
+
−
Spectrophotometer</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l9 level1 lfo19'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Pipette 1-2µl of blanking buffer onto the bottom
+
−
pedestal, lower the arm and click the BLANK button. The blank solution is the
+
−
same buffer that the DNA of interest is dissolved in.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddle style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l9 level1 lfo19'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Wipe away the
+
−
blank. Pipette 1-2µl of DNA sample and hit MEASURE<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l9 level1 lfo19'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Wipe away the
+
−
last sample to prepare for the next sample, and repeat.<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.df10up6a0vzh></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>PCR Mixture: General recipe for PCR is as follows:</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l11 level1 lfo20'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>4 µL of 5x <span class=SpellE>phusion</span> buffer.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l11 level1 lfo20'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>0.4 µL <span class=SpellE>dNTPs</span>.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l11 level1 lfo20'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>0.6 µL of DMSO.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l11 level1 lfo20'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:13.5pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>0.2 µL of polymerase</span><span style='font-size:
+
−
13.5pt'>.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l11 level1 lfo20'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>11.8 µL of water.<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.6eeyqdi9mhqi></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>To this mixture, add:</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l8 level1 lfo21'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>1 µL of forward primer.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l8 level1 lfo21'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>1 µL of reverse primer.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l8 level1 lfo21'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>1 µL of template.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l8 level1 lfo21'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Total volume = 20 µL<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.ka6u2m955lhu></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>PCR settings: A general program for PCR is as follows:</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l4 level1 lfo22'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Initial <span class=SpellE>denaturation</span> at 98<sup>o</sup>C
+
−
for 30seconds.<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.xq8hqhbk6lk5></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>Followed by 30 repeats of:</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:59.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l32 level1 lfo23'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
class=SpellE><span style='font-size:14.0pt'>Denaturation</span></span><span
+
−
style='font-size:14.0pt'> &#8211; 98<sup>o</sup>C for 10seconds.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l32 level1 lfo23'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Annealing &#8211; 60<sup>o</sup>C for 10seconds.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:59.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l32 level1 lfo23'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Extension &#8211; 72<sup>o</sup>C for 2minutes.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:59.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l32 level1 lfo23'><![if !supportLists]><span
+
−
style='font-size:12.0pt;mso-bidi-font-size:14.0pt;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial'><span style='mso-list:Ignore'>&#9679;<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Final extension &#8211; 72<sup>o</sup>C for 10minutes.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt'>Plasmid Prep<o:p></o:p></span></b></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge
+
−
@13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL <span
+
−
class=SpellE>Eppendorf’s</span><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Discard
+
−
supernatant and add 1.9 <span class=SpellE>mL</span> of culture and centrifuge
+
−
again @13,200 rpm for 10 min<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Re-suspend <span
+
−
class=SpellE>pelleted</span> cells in P1 Buffer (250µl) <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Add 250µL of P2
+
−
buffer &amp; invert 4-6 times (turns homogenous blue)<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Add 350µL of N3
+
−
&amp; mix by inverting (turns colourless)<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge @
+
−
10min 13,000 rpm to obtain a pellet<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Transfer
+
−
supernatant in QIA Prep Spin Column by <span class=SpellE>pipetting</span><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>8.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge 30-60
+
−
sec - discard flow through<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>9.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Wash QIA Prep
+
−
Spin Column with 0.5mL of PB<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>10.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge for
+
−
30-60 seconds - discard flow through<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>11.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Wash spin column
+
−
by adding 0.75 <span class=SpellE>mL</span> PE buffer<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>12.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge for
+
−
30-60 seconds - discard flow through and centrifuge for a further 1min<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>13.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Place <span
+
−
class=SpellE>QIAPrep</span> Column in clean <span class=SpellE>Eppendorf</span><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>14.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Elute DNA add
+
−
50µl of water, stand for 1min, centrifuge for 1min<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l29 level1 lfo24'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>15.<span style='font:7.0pt "Times New Roman"'>
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>QIA prep - Spin <span
+
−
class=SpellE>miniprep</span> buffer<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><o:p>&nbsp;</o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt'>SDS-PAGE<o:p></o:p></span></b></p>
+
−
 
+
−
<p class=MsoNormal><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Re-suspend <span
+
−
class=SpellE>pelleted</span> bacterial cells in 200µL of <span class=SpellE>Milli</span>-Q-water<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Transfer 50 µL of
+
−
suspension into new <span class=SpellE>Eppendorf</span> tubes and combine with
+
−
50ul of 2xTruSep sample buffer.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'><span
+
−
style="mso-spacerun: yes">&nbsp;</span>Shear the cells using a Hamilton
+
−
syringe.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Centrifuge the
+
−
preparation for 3minutes @ 13,000 rpm.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Load 20 µL of the
+
−
supernatant into gel.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Conduct
+
−
electrophoresis at a constant voltage (200V) for 1 hour.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l21 level1 lfo25'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span class=SpellE><span style='font-size:14.0pt'>Coommassie</span></span><span
+
−
style='font-size:14.0pt'> Stain for ~30 minutes.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt'>Heat Shock<o:p></o:p></span></b></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Obtain competent
+
−
cells from -80<sup>o</sup>C.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Defrost gently on
+
−
ice. 100µl is sufficient for 2 transformations.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Add 1-10µl of
+
−
plasmid DNA/ ligation mix to each tube. Incubate on ice for 5 min.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Put the tubes in
+
−
the 42<sup>o</sup>C water bath for 30 seconds, <span class=GramE>then</span>
+
−
back on ice for 2 min.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Add 200µl of SOC
+
−
media to each tube, and incubate in the 37<sup>o</sup>C shaker for 10 min or up
+
−
to 1h (10min for purified plasmid or 1h for ligation mix).<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l31 level1 lfo26'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>For each tube of
+
−
cells, spread 50µl onto one LB plate with appropriate antibiotic, and 500µl
+
−
onto a second plate, using aseptic technique. Place your plate upside-down in
+
−
the 37<sup>o</sup>C incubator.<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:normal'><span
+
−
style='font-size:14.0pt'><o:p>&nbsp;</o:p></span></p>
+
−
 
+
−
<h4 style='line-height:160%;mso-pagination:widow-orphan;page-break-after:auto'><a
+
−
name=h.uwg4fet686y3></a><b style='mso-bidi-font-weight:normal'><span
+
−
style='font-size:15.0pt;line-height:160%;font-family:Arial;mso-fareast-font-family:
+
−
Arial;mso-bidi-font-family:Arial;color:#333333;text-decoration:none;text-underline:
+
−
none'>Induction of <span class=SpellE>Operons</span></span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-align:
+
−
justify;text-indent:-18.0pt;line-height:normal;mso-list:l13 level1 lfo27'><![if !supportLists]><span
+
−
style='font-size:14.0pt'><span style='mso-list:Ignore'>1.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt'>Re-transform restriction-digest screened plasmids
+
−
according to the heat shock transformation protocol.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l13 level1 lfo27'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Screen plates for
+
−
transformant colonies and place in 5mL of ZYM-5052 broth with appropriate
+
−
antibiotic concentration at 37<sup>o</sup>C with shaking until OD600¬ is 2.0
+
−
&#8211; 3.0.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
auto;text-align:justify;text-indent:-18.0pt;mso-list:l13 level1 lfo27'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Take whole volume
+
−
and place in 45mL of ZYM-5052 broth in a sterile conical flask containing
+
−
appropriate antibiotic concentration. Incubate at 37<sup>o</sup>C with shaking.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l13 level1 lfo27'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Cell pellets were
+
−
collected through centrifugation at 12000 rpm for 5 mins. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxsplast style='margin-left:36.0pt;mso-add-space:auto;
+
−
text-align:justify;text-indent:-18.0pt;mso-list:l13 level1 lfo27'><![if !supportLists]><span
+
−
style='font-size:14.0pt;mso-fareast-font-family:Times;mso-bidi-font-family:
+
−
Times'><span style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Cell pellets were
+
−
resuspended in re-suspension buffer (Containing: 10% glycerol w/v, 50mM <span
+
−
class=SpellE>Tricene</span> <span class=SpellE>NaOH</span> pH 8.0, 2mM MgCl<sub>2</sub>,
+
−
1mM DTT) and whole lysate was collected through a French Press for functional
+
−
assays of <span class=SpellE>operon</span> protein products in lysate.<o:p></o:p></span></p>
+
−
 
+
−
<h4 style='text-align:justify;line-height:160%;mso-pagination:widow-orphan;
+
−
page-break-after:auto'><a name=h.s0f83usywiy1></a><span class=SpellE><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt;line-height:
+
−
160%;font-family:Arial;mso-fareast-font-family:Arial;mso-bidi-font-family:Arial;
+
−
color:#333333;text-decoration:none;text-underline:none'>MgPE</span></b></span><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt;line-height:
+
−
160%;font-family:Arial;mso-fareast-font-family:Arial;mso-bidi-font-family:Arial;
+
−
color:#333333;text-decoration:none;text-underline:none'> enzymatic functional
+
−
assay</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='margin-left:36.0pt;mso-add-space:auto;text-indent:
+
−
-18.0pt;mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:
+
−
14.0pt;line-height:115%'><span style='mso-list:Ignore'>1.<span
+
−
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
style='font-size:14.0pt;line-height:115%'>Collect whole lysate, from induced <span
+
−
class=SpellE>operon</span> 3 containing <span class=SpellE>ChlM</span>
+
−
expressed proteins, using <span class=SpellE>ChlM</span> <span class=SpellE>pET</span>
+
−
(L. <span class=SpellE>Meinecke</span> et <span class=GramE>al.2010)as</span> a
+
−
negative control.<span style="mso-spacerun: yes">&nbsp; </span><o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpFirst style='margin-top:.1pt;margin-right:0cm;
+
−
margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt;
+
−
mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt;
+
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mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span
+
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style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Prepare a master
+
−
mix of recombinant <span class=SpellE>ChlM</span>, 25µl of S-<span
+
−
class=SpellE>Adenosyl</span>-L- <span class=SpellE>methionine</span> and 65µl
+
−
of 2µM of Mg-<span class=SpellE>protoporphyrin</span> IX.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-top:.1pt;margin-right:0cm;
+
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margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt;
+
−
mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt;
+
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mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span
+
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style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
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</span></span></span><![endif]><span style='font-size:14.0pt'>Incubate at 30</span><span
+
−
style='font-size:14.0pt;font-family:"Noteworthy Light";mso-bidi-font-family:
+
−
"Noteworthy Light"'>&#8451;</span><span style='font-size:14.0pt'> with gentle
+
−
agitation for 8-10min<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-top:.1pt;margin-right:0cm;
+
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margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt;
+
−
mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt;
+
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mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span
+
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style='mso-list:Ignore'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>100µL of acetone
+
−
was added to precipitate protein. <o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpMiddle style='margin-top:.1pt;margin-right:0cm;
+
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margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt;
+
−
mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt;
+
−
mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span
+
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style='mso-list:Ignore'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>The sample was
+
−
spun down, to separate the protein and supernatant.<o:p></o:p></span></p>
+
−
 
+
−
<p class=normalcxspmiddleCxSpLast style='margin-top:.1pt;margin-right:0cm;
+
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margin-bottom:.1pt;margin-left:36.0pt;mso-add-space:auto;text-indent:-18.0pt;
+
−
mso-list:l2 level1 lfo28'><![if !supportLists]><span style='font-size:14.0pt;
+
−
mso-fareast-font-family:Times;mso-bidi-font-family:Times'><span
+
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style='mso-list:Ignore'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
+
−
</span></span></span><![endif]><span style='font-size:14.0pt'>Supernatant was
+
−
then subjected to UHPLC to and observed peaks. <o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal><span style='font-size:14.0pt;line-height:115%'><o:p>&nbsp;</o:p></span></p>
+
−
 
+
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<p class=MsoNormal><span style='font-size:14.0pt;line-height:115%'><span
+
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style="mso-spacerun: yes">&nbsp;</span></span></p>
+
−
 
+
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<h4 style='text-align:justify;line-height:160%;mso-pagination:widow-orphan;
+
−
page-break-after:auto'><a name=h.ynrtu1oxkw8d></a><b style='mso-bidi-font-weight:
+
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normal'><span style='font-size:15.0pt;line-height:160%;font-family:Arial;
+
−
mso-fareast-font-family:Arial;mso-bidi-font-family:Arial;color:#333333;
+
−
text-decoration:none;text-underline:none'>UHPLC protocol</span></b></h4>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
style='font-size:14.0pt;line-height:160%'>The UHPLC protocol was performed
+
−
according to <span class=SpellE>Sawicki</span> and Willows (2007). Briefly, a
+
−
Shimadzu HPLC system was used at 2ml/min with an <span class=SpellE>Alltech</span>
+
−
C8 column. A 5 min linear gradient was used for separation of Mg-<span
+
−
class=SpellE>protoporphyrin</span> IX and Magnesium-<span class=SpellE>protoporphyrin</span>
+
−
IX mono-methyl ester. Compounds were detected at an excitation wavelength of
+
−
excitation of 416nm and emission of 595nm.<span style="mso-spacerun:
+
−
yes">&nbsp; </span><o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:160%'><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt;line-height:
+
−
160%'><o:p>&nbsp;</o:p></span></b></p>
+
−
 
+
−
<p class=MsoNormal style='text-align:justify;line-height:160%'><b
+
−
style='mso-bidi-font-weight:normal'><span style='font-size:15.0pt;line-height:
+
−
160%'>Auto induction recipe <o:p></o:p></span></b></p>
+
−
 
+
−
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+
−
style='border-collapse:collapse;border:none;mso-border-alt:solid black;
+
−
mso-border-themecolor:text1;mso-border-alt:.5pt;mso-yfti-tbllook:191;
+
−
mso-padding-alt:0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid black;
+
−
mso-border-insideh-themecolor:text1;mso-border-insidev:.5pt solid black;
+
−
mso-border-insidev-themecolor:text1'>
+
−
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
+
−
  <td width=239 valign=top style='width:239.4pt;border:solid white;mso-border-themecolor:
+
−
  background1;border:1.0pt;border-bottom:none;mso-border-top-alt:solid white;
+
−
  mso-border-top-themecolor:background1;mso-border-top-alt:.5pt;mso-border-left-alt:
+
−
  solid white;mso-border-left-themecolor:background1;mso-border-left-alt:.5pt;
+
−
  mso-border-right-alt:solid white;mso-border-right-themecolor:background1;
+
−
  mso-border-right-alt:.5pt;background:#FEFEFE;mso-shading:windowtext;
+
−
  mso-pattern:solid white;padding:0cm 5.4pt 0cm 5.4pt'>
+
−
  <p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
  style='font-size:14.0pt;line-height:160%'>1.ZYM-5052 Medium <o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l1 level1 lfo29'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>ZY media 9.57ml <o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l1 level1 lfo29'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>1m MgSO4 20ul<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l1 level1 lfo29'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>50x5052 200ul<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l1 level1 lfo29'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>50 x M 200ul<o:p></o:p></span></p>
+
−
  <p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
  style='font-size:15.0pt;line-height:160%'><o:p>&nbsp;</o:p></span></p>
+
−
  </td>
+
−
  <td width=239 valign=top style='width:239.4pt;border:solid white;mso-border-themecolor:
+
−
  background1;border:1.0pt;border-left:none;mso-border-left-alt:solid white;
+
−
  mso-border-left-themecolor:background1;mso-border-left-alt:.5pt;mso-border-alt:
+
−
  solid white;mso-border-themecolor:background1;mso-border-alt:.5pt;padding:
+
−
  0cm 5.4pt 0cm 5.4pt'>
+
−
  <p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
  style='font-size:14.0pt;line-height:160%'>2. 50 x M<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>DI water 80ml<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>Na2HPO4 17.75g<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>KH2PO4 17g<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>NH4Cl 13.4<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l25 level1 lfo30'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>Na2SO4 3.55g<o:p></o:p></span></p>
+
−
  <p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
  style='font-size:15.0pt;line-height:160%'><o:p>&nbsp;</o:p></span></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:1;mso-yfti-lastrow:yes'>
+
−
  <td width=239 valign=top style='width:239.4pt;border:solid white;mso-border-themecolor:
+
−
  background1;border:1.0pt;border-top:none;mso-border-left-alt:solid white;
+
−
  mso-border-left-themecolor:background1;mso-border-left-alt:.5pt;mso-border-bottom-alt:
+
−
  solid white;mso-border-bottom-themecolor:background1;mso-border-bottom-alt:
+
−
  .5pt;mso-border-right-alt:solid white;mso-border-right-themecolor:background1;
+
−
  mso-border-right-alt:.5pt;padding:0cm 5.4pt 0cm 5.4pt'>
+
−
  <p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
  style='font-size:14.0pt;line-height:160%'>3. 50 x 5052<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l6 level1 lfo31'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>Glycerol 25g (weigh in Beaker)<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l6 level1 lfo31'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>DI water 73ml<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l6 level1 lfo31'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>Glucose 2.5g<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpLast style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l6 level1 lfo31'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  class=GramE><span style='font-size:14.0pt;line-height:160%'>alpha</span></span><span
+
−
  style='font-size:14.0pt;line-height:160%'> lactose 10g<o:p></o:p></span></p>
+
−
  <p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
  style='font-size:15.0pt;line-height:160%'><o:p>&nbsp;</o:p></span></p>
+
−
  </td>
+
−
  <td width=239 valign=top style='width:239.4pt;border-top:none;border-left:
+
−
  none;border-bottom:solid white;mso-border-bottom-themecolor:background1;
+
−
  border-bottom:1.0pt;border-right:solid white;mso-border-right-themecolor:
+
−
  background1;border-right:1.0pt;mso-border-top-alt:solid white;mso-border-top-themecolor:
+
−
  background1;mso-border-top-alt:.5pt;mso-border-left-alt:solid white;
+
−
  mso-border-left-themecolor:background1;mso-border-left-alt:.5pt;mso-border-alt:
+
−
  solid white;mso-border-themecolor:background1;mso-border-alt:.5pt;padding:
+
−
  0cm 5.4pt 0cm 5.4pt'>
+
−
  <p class=MsoNormal style='text-align:justify;line-height:160%'><span
+
−
  style='font-size:14.0pt;line-height:160%'>4. 1M MgSO4<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpFirst style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l26 level1 lfo32'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>MgSO4.7H2O 24.65g<o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l26 level1 lfo32'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
+
−
  mso-bidi-font-family:Times'><span style='mso-list:Ignore'>&#9679;<span
+
−
  style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
−
  style='font-size:14.0pt;line-height:160%'>DI water 87ml <o:p></o:p></span></p>
+
−
  <p class=normalcxspmiddleCxSpMiddle style='margin-left:36.0pt;mso-add-space:
+
−
  auto;text-align:justify;text-indent:-18.0pt;line-height:160%;mso-list:l26 level1 lfo32'><![if !supportLists]><span
+
−
  style='font-size:14.0pt;line-height:160%;mso-fareast-font-family:Times;
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Latest revision as of 03:52, 19 September 2015

Experiments and Protocol
Project Description page
Link to Experiments & Protocols page
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Fig 1. An overview of the methodology. A modified BioBrick Standard Assembly was used. Alkaline phosphatase on the target vector backbone for the genes; ChlM and those from Photosystem II. The Gibson Assembly method was used in order to insert composite part ChlH into the BioBrick vector plasmid.

Experiments and Protocol

Making Competent Cells

  1. Using a sterile plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22oC, 200-250rpm.
  2. A600 should be 0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5.
  3. Remove the flask from the incubator and place on ice for 10 minutes.
  4. Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4oC
  5. Pour off and discard the supernatant, and immediately place the tube on ice.
  6. Resuspend your cells in 1mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.
  7. Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30mL in this case). Mix the tube by gently inverting 3 times.
  8. Incubate the tube on ice for 10 minutes.
  9. Centrifuge at 2,500 x g for 7 minutes at 4oC, discard the supernatant.
  10. Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. e.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.
    11. Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.
  11. Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.
  12. Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen.
  13. Repeat step 11-13 for the rest of your cell suspension in step 10. Store cells at -80oC
TB Buffer
Ingredients: 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl.
Methods: All components (except for MnCl2-4H2O) were mixed and dissolved in 500 mL of water and pH adjusted to 6.7 with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL of water, mixed and solution adjusted to 1 L. Sterilisation via filtration followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.
SOB Media (for competent cells)
Ingredients: 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl NaCl, 417µL KCl, 1.205g Mg2SO4 & 500mL Milli-Q water.
Methods: The adjusted quantities were combined in a 1 L measuring column with constant stirring and then placed in the autoclave for sterilisation (121oC, 15 min, standard liquid cycle).

Agarose Gel Electrophoresis

  1. Mix 1ul of 1kbp DNA ladder with 6ul of loading dye (bromophenol blue) and 4ul of 1x TAE buffer and load into first well
  2. Mix 5ul of plasmid DNA with 1ul of loading dye and load into wells.
  3. Run gel at 90V for 45minutes approximately
  4. Gel image is taken using Biorad GelDoc
EDTA Buffer
Ingredients: 37.22g EDTA solid, 180 mL of water and pH adjusted to 8.0 using 10M NaOH.
Methods: Components were combined then pH adjusted.
50 X TAE Buffer
Ingredients: 121.2g Tris base (dissolved in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0).
Methods: Components were combined then make up to 500ml with distilled water.
Making Agarose Gel
Ingredients: 1g agarose powder, 100ml of 1 x TAE
Methods: Dissolve agarose in TAE by microwaving for 1 minute, wait till cool to touch, then add 2ul of GelRed and pour into the casette.

Composite Part Ligation

The assembly of composite parts from two existing BioBricks i.e. BioBrick plasmid A (parent vector) and BioBrick plasmid B (gene to be inserted) was performed through a restriction digest/ligation protocol.

  1. 200ng of each BioBrick plasmid was digested as follows: Plasmid BioBrick A with NEB restriction enzymes SpeI and PstI; plasmid BioBrick B with XbaI and PstI, according to supplier protocol. (1hr @37oC, then 20mins @80oC).
  2. 1µL of Fast alkaline phosphatase (Thermo Scientific) was added to reaction tube of plasmid BioBrick A to dephosphorylate the gene fragments and prevent re-ligation of the parent vector. Reaction tubes were incubated at 37oC for 60 mins, followed by a deactivation step at 80oC for 20 mins.
  3. Ligation was then performed with an insert to vector molar ratio of 3:1. 1µL of T4 ligase (NEB) was added to the mix for ligation, according to supplier protocol. Ligation reactions were performed at 37oC for 60 mins and kept on ice for transformation into chemical competent cells.


Plasmid Prep

  1. Centrifuge @13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorf’s
  2. Discard supernatant and add 1.9 mL of culture and centrifuge again @13,200 rpm for 10 min
  3. Re-suspend pelleted cells in P1 Buffer (250µl)
  4. Add 250µL of P2 buffer & invert 4-6 times (turns homogenous blue)
  5. Add 350µL of N3 & mix by inverting (turns colourless)
  6. Centrifuge @ 10min 13,000 rpm to obtain a pellet
  7. Transfer supernatant in QIA Prep Spin Column by pipetting
  8. Centrifuge 30-60 sec - discard flow through
  9. Wash QIA Prep Spin Column with 0.5mL of PB
  10. Centrifuge for 30-60 seconds - discard flow through
  11. Wash spin column by adding 0.75 mL PE buffer
  12. Centrifuge for 30-60 seconds - discard flow through and centrifuge for a further 1min
  13. Place QIAPrep Column in clean Eppendorf
  14. Elute DNA by adding 30µl of water, stand for 1min, centrifuge for 1min
  15. Plasmid concentration is measured using NanoDrop 2000 spectrophotometer


General PCR Recipe

  • 4 µL of 5x phusion buffer.
  • 0.4 µL dNTPs.
  • 0.6 µL of DMSO.
  • 0.2 µL of polymerase.
  • 11.8 µL of water.
To this mixture, add:
  • 1 µL of forward primer.
  • 1 µL of reverse primer.
  • 1 µL of template.
  • Total volume = 20 µL



PCR settings: A general program for PCR is as follows:

  • Initial denaturation at 98oC for 30seconds.
Followed by 30 repeats of:
  • Denaturation – 98oC for 10seconds.
  • Annealing – 60oC for 10seconds.
  • Extension – 72oC for 2minutes.
  • Final extension – 72oC for 10minutes.


Transformation By Heat Shock

  1. Obtain competent cells from -80oC.
  2. Defrost gently on ice. 100µl is sufficient for 2 transformations.
  3. Add 1-10µl of plasmid DNA/ ligation mix to each tube. Incubate on ice for 30 min.
  4. Heat shock in 42oC water bath for 30 seconds, then back on ice for 2 min.
  5. Add 200µl of SOC media to each tube, and incubate in the 37oC shaker for 1h
  6. For each tube of cells, spread 50µl onto one LB plate with appropriate antibiotic, and 100µl onto a second plate, using aseptic technique. Place your plate upside-down in the 37oC incubator.
LB Agar Plate Preparation
Ingredients: 15g Bacto agar, 1000mL LB media
Methods: Dissolve agar in LB media, autoclave for 15 mins at 121oC; wait until cool to touch, then add appropriate antibiotics before pouring into petri dishes.
LB Media
Ingredients: Tryptone 10g, Yeast extract 5g, NaCl 10g, Milli-Q water to 1000mL.
Methods: Dissolved 10g tryptone, 5g yeast extract and 10g NaCl in 800mL Milli-Q water, making use of a magnetic stirrer. Once dissolved, bring volume up to 1 L using Milli-Q water. Autoclave 1L of the solution (121oC, 15 min, standard liquid cycle).
SOC Media
Ingredients: 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl 5M NaCl, 417µL 3M KCl, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose & 500mL Milli-Q water.
Methods: The adjusted quantities were combined in a 1 L measuring column with constant stirring and then placed in the autoclave for sterilisation (121oC, 15 min, standard liquid cycle).

Protein Expression

  1. Re-transform restriction-digest screened plasmids according to the heat shock transformation protocol.
  2. Screen plates for transformant colonies and place in 5mL of ZYM-5052 broth with appropriate antibiotic concentration at 37oC with shaking until OD600¬ is 2.0 – 3.0.
  3. Take whole volume and place in 45mL of ZYM-5052 broth(1) in a sterile conical flask containing appropriate antibiotic concentration. Incubate at 37oC with shaking.
  4. Cell pellets were collected through centrifugation at 12000 rpm for 5 mins.
  5. Cell pellets were resuspended in re-suspension buffer (Containing: 50mM Tricine NaOH pH 8.0, 2mM MgCl2, 1mM DTT) and whole lysate was collected through a French Press for functional assays of operon protein products in lysate.
Auto induction Medium (ZYM-5052)
  • ZY media 9.57ml
  • 1m MgSO4 20ul
  • 50x5052 200ul
  • 50 x M 200ul
1. 50 x M
  • DI water 80ml
  • Na2HPO4 17.75g
  • KH2PO4 17g
  • NH4Cl 13.4
  • Na2SO4 3.55g
2. 50 x 5052
  • Glycerol 25g (weigh in Beaker)
  • DI water 73ml
  • Glucose 2.5g
  • alpha lactose 10g
3. 1M MgSO4
  • MgSO4.7H2O 24.65g
  • DI water 87ml
4. ZY Media (400mL)
  • Trypton 4g
  • Yeast extract 2g


MgPE Enzymatic Functional Assay

  1. Collect whole lysate, from induced operon 3 containing ChlM expressed proteins, using ChlM pET(2) as a negative control.
  2. Prepare a master mix of recombinant ChlM, 25µl of S-Adenosyl-L- methionine and 65µl of 2µM of Mg-protoporphyrin IX.
  3. Incubate at 30℃ with gentle agitation for 8-10min
  4. 100µL of acetone was added to precipitate protein.
  5. The sample was spun down, to separate the protein and supernatant.
  6. Supernatant was then subjected to UHPLC to and observed peaks.


UHPLC

The UHPLC was performed according to Sawicki and Willows(3). Briefly, a Shimadzu HPLC system was used at 2ml/min with an Alltech C8 column. A 5 min linear gradient was used for separation of Mg-protoporphyrin IX and Magnesium-protoporphyrin IX mono-methyl ester. Compounds were detected at an excitation wavelength of excitation of 416nm and emission of 595nm.



SDS-PAGE Electrophoresis

  1. Re-suspend pelleted bacterial cells in 200µL of Milli-Q-water
  2. Transfer 50 µL of suspension into new Eppendorf tubes and combine with 50ul of 2xTruSep sample buffer.
  3. Shear the cells using a Hamilton syringe.
  4. Centrifuge the preparation for 3minutes @ 13,000 rpm.
  5. Load 20 µL of the supernatant into gel.
  6. Conduct electrophoresis at a constant voltage (200V) for 1 hour.
  7. Coommassie Stain for ~30 minutes.


Gun4 PPIX binding by fluorescence spectrophotometry

Preparation of PPIX
  1. Approximately 1mg of dried PPIX was dissolved in 5ul of 1M NaOH, and diluted with 200ul of deionized water.
  2. Pellet any undissolved particulate by centrifugation, and dilute the supernatant 100-fold with 5% HCl.
  3. The concentration of the PPIX solution was calculated by measuring the absorbance of the diluted solution at 404nm, using the Beer-Lambert equation with molar extinction coefficient of 278000.
  4. A 2uM concentration solution of PPIX was used for the binding assay.
Gun4 PPIX binding
  1. Over-expressed Gun4 containing cell lysates was prepared as described above.
  2. The protein concentration was estimated using Bradford reagent, and the lysate diluted to approximately 0.2mg/ml.
  3. 100ul of the 2uM PPIX solution was added with varying amounts of protein lysate to a total volume of 200ul, and transferred to a clean quartz cuvette for fluorescence measurements in a Cory Eclipse fluorescence spectrophotometer (Agilent)
  4. The excitation wavelength was set at 280nm and the emission at measure from 580nm to 800nm. The emission maxima for protein bound PPIX is at 635nm.


References
  1. Studier, F. W. (2014). Stable expression clones and auto-induction for protein production in E. coli. In Structural Genomics (pp. 17-32). Humana Press.
  2. Meinecke, L., Alawady, A., Schroda, M., Willows, R., Kobayashi, M. C., Niyogi, K. K., ... & Beck, C. F. (2010). Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX. Plant molecular biology, 72(6), 643-658.
  3. Sawicki, A., & Willows, R. (2007). S-Adenosyl-L-methionine: magnesium-protoporphyrin IX O-methyltransferase from Rhodobacter capsulatus: mechanistic insights and stimulation with phospholipids. Biochem. J, 406, 469-478.