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                            <h1 id="back_to_the_top">October 10th 2016</h1>
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                                     <h2 class="blog_topHd"> <font color =”#279AD3”>PCR on G3</font></h2> 
                                  </div>         
                                   
              <h3><font color =”94FAF1”> Objectives </font></h3>                       
             <p>The overall purpose is to amplify DNA fragment for further digestions and ligations in order to construct biobrick:</p>
                  <li><p>G3 : Gluc optimised —>  isolation of Gluc from BBB to create BBG3</p></li>

                        <h3><font color =”94FAF1”> Materials </font></h3>
<p>DNA fragments: BBB-4 (from mini prep 22/09/16)<br/>
Primers: Fw-Gluc (forward) and Rev-Gluc (reverse)</p>

                                  <h3><font color =”94FAF1”> Protocol </font></h3>    
                                     
        <h5><font color =”#3CB5E1”>PCR</font></h5> 

<p>1.  Mix for 2 samples (Total volume of Mix : 96 µL), in an Eppendorf tube :</p>

<li><p>79.5 µL H2O</p></li>
<li><p>10 µL Buffer Taq (1 X final, NEB #B9014S)</p></li>
<li><p>2 µL dNTP (200 µM final, NEB #N0447S)</p></li>
<li><p>2 µL Primer Fw-Gluc</p></li>
<li><p>2 µL Primer Rev-Gluc</p></li>
<li><p>0.5 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S)</p></li>

<p>2.  Add in 2 PCR tubes, in the respected order:</p>

<li><p>48 µL Mix</p></li>
<li><p>2 µL of DNA fragment  (BBB) or 2 µL H20 (Control)</p></li>
<p>—> Gently mix the reaction and short spin centrifugation</p>

<p>3.  Set the following parameters for the PCR reaction :</p>

<li><p>G3 (617 bp):<br/>
     Lid: 95°C<br/>
     Initial denaturation: 95°C, 30 s<br/> 
     30 cycles of: 95°C, 30 s<br/>
                          50°C, 1 min<br/>
                          68°C, 37 s<br/>
     Final extension: 68°C, 5 min<br/>
     Hold: 4°C</p></li>

<h5><font color =”#3CB5E1”>Electrophoresis: for screening the PCR results</font></h5> 

<p>1% Agarose gel:</p>
  <ol><li><p>Put 1 g of agarose + 100 mL of TAE 1X in a bottle of 500 mL.</p></li>
          <li><p>Mix and heat it 2 min 30 s in the microwaves. Wait the cooling of the bottle until it is tepid.</p></li>
          <li><p>Add 5 µL of Gel Red 10,000 X (0.5 X final).</p></li>
          <li><p>Flow the gel and place the combs.</p></li>
          <li><p>Wait until it is solidified. Remove slowly the combs.</p></li>  </ol>
   
<p>Drop-off:</p>
<ol><li><p>Short Speed centrifugation of samples.</p></li>
<li><p>Addition of 2 µL of Purple loading dye 6 X in 10 µL of sample.</p></li>
<li><p>Drop-off 10 µL of Purple ladder and 12 µL of each sample (G3: PCR product, CG3: PCR purified G3).</p></li>

                     <figure>
                                    <center><img src="https://static.igem.org/mediawiki/2016/8/82/T--Ionis_Paris--Notebook10_10Fig.1.png" alt=""></center>
                                </figure>

<li><p>Run at 90 V</p></li></ol>

<h5><font color =”#3CB5E1”>PCR purification :</font></h5> 

                               <p>QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available <a href="https://www.qiagen.com/fi/resources/resourcedetail?id=390a728a-e6fc-43f7-bf59-b12091cc4380&lang=en">here</a>)</p>

<ol>
<li><p>Add 5 volumes Buffer PB (250 µL) to 1 volume of the PCR reaction (50 µL) and mix. The color of the mixture is yellow.</p></li>
<li><p>Load the sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through.</p></li>  
<li><p>Add 750 µL Buffer. Centrifuge for 1 min at 13,000 rpm and discard flow-through.</p></li>
<li><p>Centrifuge once more for 1 min at 13,000 rpm.</p></li>
<li><p>Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.</p></li>
<li><p>Add 50 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.</p></li> 
<li><p>Store the purified DNA at -20°C.</p></li></ol>

                                  <h3><font color =”94FAF1”> Results</font></h3>    

<h5><font color =”#3CB5E1”>Electrophoresis:</font></h5> 
<p><font color= ”46BB0A”>Expected results / Obtained results:</font></p>

                     <figure>
                                    <center><img src="https://static.igem.org/mediawiki/2016/8/8c/T--Ionis_Paris--Notebook10_10Fig.2.png" alt=""></center>
                                </figure>

                                  <h3><font color =”94FAF1”>Interpretation</font></h3>    

<p>We obtain the desired strip for G3, as shown on the gel above the strips are closed to 617 bp.<br/>
It seems that G3 has been properly amplified.</p>


                         <h2 class="blog_topHd"> <font color =”#279AD3”>Miniprep: on DH5⍺ transformed with BBG1</font></h2> 
                                                                      
                                    <h3><font color =”94FAF1”> Objectives </font></h3>                       
             <p>Purification of BBG1 plasmid extracted from bacterial mini-cultures in order to have a stock of plasmid.</p>

                                   <h3><font color =”94FAF1”> Materials </font></h3>
                                          
<P>Mini-culture of bacteria transformed with BBG1 realized the 09/10 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube).<br/>
From those mini-cultures, take 500 µL to realize a glycerol stock of tranformed bacteria. The 4.5 mL remaining will serve for the miniprep.</p>
                               
                                 <h3><font color =”94FAF1”>Protocol</font></h3>

                               <p>The 4 miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104)  and following the protocol given by the supplier  (available <a href="https://www.qiagen.com/fr/resources/resourcedetail?id=331740ca-077f-4ddd-9e5a-2083f98eebd5&lang=en">here</a>)</p>

                      <h5><font color =”#3CB5E1”>Miniprep: </font></h5> 
 <ol>                     
<li><p>Divide each 4.5 mL bacterial O/N mini-cultures into 4 Eppendorf tubes and centrifuge all those tubes at 9,000 rpm for 3 min at room temperature. Discard the supernatant.</p></li>
<li><p>Resuspend the pellet in 62.5 μL Buffer P1 and pool the 4 Eppendorf tubes into a unique tube.</p></li>
<li><p>Add 250 μL Buffer P2 and mix by inverting the tube 6 times. The solution turns blue.</p></li> 
<li><p>Add 350 μL Buffer N3 and mix by inverting the tube 6 times. The solution turns colorless.</p></li>
<li><p>Centrifuge for 10 min at 13,000 rpm.</p></li>
<li><p>Load 800 μL supernatant from step 5 to the QIAprep 2.0 spin column. Centrifuge for 1 min and discard the flow-through.</p></li>
<li><p>Add 500 µL Buffer PB. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.</p></li>
<li><p>Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.</p></li>
<li><p>Centrifuge once more for 1 min at 13,000 rpm.</p></li>
<li><p>Place the QIAprep 2.0 spin column in a clean 1.5 mL microcentrifuge tube.</p></li>
<li><p>Add 50 μL Buffer EB to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.</p></li>
<li><p>Store the purified DNA at -20°C.</p></li>
</ol>

                      <h5><font color =”#3CB5E1”>Bacteria storage : </font></h5> 

      <p>1.  Add 100 µL of glycerol 50% to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf.</p>
      <p>12 tubes of BBG1 (3 per mini-cultures)</p>
<p>2.  Store at -80°C.</p>
                        
                         <h2 class="blog_topHd"> <font color =”#279AD3”>Digestion: C4 and pSB1A2-C5; BB12 and BBX4; P12His and BB-B0015; G3 and pSB1C3</font></h2> 

                                         <h3><font color =”94FAF1”>Objective</font></h3>
<p>
Double digestion of C4 by EcoRI and SpeI and of pSB1A2-C5 by EcoRI and XbaI for the subsequent ligation of C4 in pSB1A2-C5 to obtain C45.<br/>
Double digestion of BB12 by SpeI and PstI and of X4 by XbaI and PstI for the subsequent ligation of BBX4 in BB12 to obtain BBX5.<br/>
Double digestion of P12His by EcoRI and SpeI and of BB-B0015 by EcoRI and XbaI for the subsequent ligation of BB12his in term to obtain BBX3.<br/>
Double digestion of G3 by EcoRI and PstI and of pSB1C3-RFP by EcoRI and PstI for the subsequent ligation of G3 in pSB1C3 and creation of BBG3<br/></p>

                                         <h3><font color =”94FAF1”>Materials</font></h3>
                      <h5><font color =”#3CB5E1”>Stock concentrations:</font></h5> 
<p>BBC5:  ~ 150 ng/µL (from mini prep 02/10)<br/>
C4: ~ 50 ng/µL (from PCR 08/10)<br/>
BB12mut-4: ~ 250 ng/µL (from mini prep 07/09)<br/>
BBX4:  ~ 100 ng/µL (from mini prep 07/10)<br/>
P12His: ~ 50 ng/µL (from PCR 08/10)<br/>
BB-B0015: ~ 150 ng/µL (from mini prep 08/10)<br/>
G3: ~ 50 ng/µL (from PCR 10/10)<br/>
pSB1C3-RFP 4: 94 ng/µL (from mini prep 19/07)<br/></p>

                                         <h3><font color =”94FAF1”>Protocol</font></h3>
      <h5><font color =”#3CB5E1”>Digestion:</font></h5> 

<ol><li><p>In a 1.5 mL Eppendorf tube, adding in the respected order (bigger volume first and enzyme last) :</p></li>

                     <figure>
                                    <center><img src="https://static.igem.org/mediawiki/2016/7/7c/T--Ionis_Paris--Notebook10_10Fig.3.png" alt=""></center>
                                </figure>

<p>NB: The digestion were done in 20 µL.</p>

<li><p>Short Spin Centrifugation</p></li>
<li><p>Incubation 1h at 37°C</p></li>    
<li><p>Store at 4°C before purification</p></li></ol>

       <h5><font color =”#3CB5E1”>Electrophoresis for digested BBX4, pSB1C3-RFP:</font></h5> 
<p>1% Agarose gel:</p>
<ol><li><p>Put 1 g of agarose low melting point + 100 mL of TAE 1X in a bottle of 500 mL.</p></li>
<li><p>Mix and heat it 2min 30s in the microwaves. Wait the cooling of the bottle until it is tepid.</p></li>
<li><p>Add 3 µL of Gel Red 10,000X (0.3X final).</p></li>
<li><p>Flow the gel and place the combs.</p></li>
<li><p>Wait until it is solidified. Remove slowly the combs.</p></li></ol>

<p>Drop-off:</p>
<ol><li><p>Short Speed centrifugation of samples.</p></li>
<li><p>Addition of 4 µL of Purple loading dye 6X in the 20 µL of sample.</p></li>
<li><p>Drop-off 10 µL of Purple ladder and 24 µL of sample.</p></li>

                     <figure>
                                    <center><img src="https://static.igem.org/mediawiki/2016/3/3e/T--Ionis_Paris--Notebook10_10Fig.4.png" alt=""></center>
                                </figure>

<li><p>Run at 90 V.</p></li></ol>

      <h5><font color =”#3CB5E1”>Gel purification for digested X4 and pSB1C3:</font></h5> 

                               <p>QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier (available <a href="https://www.qiagen.com/us/resources/resourcedetail?id=f4ba2d24-8218-452c-ad6f-1b6f43194425&lang=en">here</a>)</p>

<ol>
<li><p>Excise the DNA fragment from the agarose gel. Gel slice Weigh = 0.342 g / 0.278 g</p></li>
<li><p>Add 3 volumes Buffer QG (1026 µL / 834 µL) to 1 volume of gel.</p></li>
<li><p>Incubate at 50°C for 10 min until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel. The color of the mixture is yellow.</p></li>
<li><p>Add 1 gel volume isopropanol to the sample and mix.</p></li>
<li><p>Load 800 µL of each samples to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Load the rest and spin again.</p></li>
<li><p>Add 500 µL Buffer QG. Centrifuge for 1 min at 13,000 rpm and discard flow-through.</p></li>
<li><p>Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through. </p></li> 
<li><p>Centrifuge once more for 1 min at 13,000 rpm.</p></li>
<li><p>Place QIAquick column into a clean 1.5 mL microcentrifuge tube.</p></li>
<li><p>Add 30 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.</p></li>
<li><p>Store the purified DNA at 4°C before the ligation.</p></li></ol>

      <h5><font color =”#3CB5E1”>PCR purification for digested P12His, BB-B0015, G3, C4, BBC5, BB12:</font></h5> 

                               <p>QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available <a href="https://www.qiagen.com/fi/resources/resourcedetail?id=390a728a-e6fc-43f7-bf59-b12091cc4380&lang=en">here</a>)</p>

<ol>
<li><p>Add 5 volumes Buffer PB (100 µL) to 1 volume of the sample (20 µL) and mix. The color of the mixture is yellow.</p></li> 
<li><p>Load the sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through.</p></li>   
<li><p>Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through.</p></li> 
<li><p>Centrifuge once more for 1 min at 13,000 rpm.</p></li> 
<li><p>Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.</p></li> 
<li><p>Add 30 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.</p></li>  
<li><p>Store the purified DNA at 4°C before the ligation.</p></li></ol>

                                  <h3><font color =”94FAF1”> Results</font></h3>    

<h5><font color =”#3CB5E1”>Electrophoresis:</font></h5> 
<p><font color= ”46BB0A”>Expected results / Obtained results:</font></p>

                     <figure>
                                    <center><img src="https://static.igem.org/mediawiki/2016/4/49/T--Ionis_Paris--Notebook10_10Fig.5.png" alt=""></center>
                                </figure>

                                  <h3><font color =”94FAF1”>Interpretation</font></h3>   

<p>The digestion of BBX4 was efficient, we get 2 strips at the end of the electrophoresis. The strip at 717 pb was the digested X4 that we purified for the subsequent ligation.<br/>
The digestion of pSB1C3-RFP was efficient, we get 2 strips at the end of the electrophoresis. The strip at 2070 pb was the digested pSB1C3 that we purified for the subsequent ligation.</p>


                         <h2 class="blog_topHd"> <font color =”#279AD3”>Ligation: P12His into BB-B0015, G3 into pSB1C3, of C4 into BBC5 and of X4 into BB12</font></h2> 

                                         <h3><font color =”94FAF1”>Objective</font></h3>

<p>Ligation of P12His into BB-B0015 to create BBX3, of G3 into pSB1C3 to create BBG3, of C4 into BBC5 to create pSB1A2-C45 and of X4 into BB12 to create X5 for subsequent transformation and creation of a stock of transformed bacteria.</p>
                               <p>The molar ratios for the ligation were calculated using NEB BioCalculator (available <a href="http://nebiocalculator.neb.com/#!/ligation">here</a>)</p>


                                         <h3><font color =”94FAF1”>Materials</font></h3>
      <h5><font color =”#3CB5E1”>Concentrations of the different components after digestion and PCR purification :</font></h5> 
<p>BB-B0015 : 5 ng/µL (150 ng into 30 µL)<br/>
P12His : 2.5 ng/µL (75 ng dans 30 µL)<br/>
G3 : 3.3 ng/µL (100 ng into 30 µL)<br/>
pSB1C3 : 2.1 ng/µL (62 ng into 30 µL)<br/>
BBC5 : 5 ng/µL (150 ng into 30 µL)<br/>
C4 : 3.3 ng/µL (100 ng into 30 µL)<br/>
BB12 : 8.3 ng/µL (250 ng into 30 µL)<br/>
X4 : 0.8 ng/µL (25 ng into 30 µL )</p>

                                         <h3><font color =”94FAF1”>Protocol</font></h3>
<ol>
<li><p>In the following order, add :</p></li>

                     <figure>
                                    <center><img src="https://static.igem.org/mediawiki/2016/0/09/T--Ionis_Paris--Notebook10_10Fig.6.png" alt=""></center>
                                </figure>

<li><p>Mix by pipetting</p></li>
<li><p>Incubate for 1h at room temperature</p></li></ol>

                         <h2 class="blog_topHd"> <font color =”#279AD3”>Transformation: competent DH5⍺ cells with ligation products BBX3, BBX5, pSB1A2-C45 and BBG3</font></h2> 
                                        
                     <h3><font color =”94FAF1”>Objective</font></h3>
<p>The objective is to transforme competent DH5⍺ cells with the ligation products BBX3, BBX5, pSB1A2-C45 and BBG3</p>

                     <h3><font color =”94FAF1”>Materials</font></h3>
<li><p>4 aliquot of 100 µL DH5⍺ competent cells (from the 20/09/16)</p></li>
<li><p>Plasmid DNA : Ligation product BBX3, BBX5, pSB1A2-C45 and BBG3</p></li>
<li><p>Petri dish LB+Cm: Cm concentration = 25 µg/mL</p></li>
<li><p>Petri dish LB+Amp: Cm concentration = 100 µg/mL</p></li>

                     <h3><font color =”94FAF1”>Materials</font></h3>

      <h5><font color =”#3CB5E1”>Experimental conditions achieved :</font></h5> 

                     <figure>
                                    <center><img src="https://static.igem.org/mediawiki/2016/b/b0/T--Ionis_Paris--Notebook10_10Fig.7.png" alt=""></center>
                                </figure>

<p>—> We need 8 LB+Cm plates+ 4 LB+Amp plates + 7 LB plates</p>

      <h5><font color =”#3CB5E1”>Transformations protocol:</font></h5
<ol>
<li><p>Thaw tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice.</p></li>
<li><p>Add the ligation products to the cell mixtures.</p></li>
<li><p>Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex.</p></li>
<li><p>Place on ice for 30 min. Do not mix.</p></li>
<li><p>Heat shock at exactly 42°C for 45 s. Do not mix.</p></li>
<li><p>Place on ice for 5 min. Do not mix.</p></li>
<li><p>Pipette 250 µL of room temperature SOC into the mixture.</p></li>
<li><p>Place at 37°C for 1h at 250 rpm.</p></li>
<li><p>Warm selection plates to 25°C.</p></li>
<li><p>Mix the cells thoroughly by flicking the tubes and inverting.</p></li>
<li><p>Spread the corresponding volume onto each plate.</p></li>
<li><p>Incubate all the plates O/N at 37°C.</p></li></ol>

                     <h3><font color =”94FAF1”>Results (obtain the 11/10)</font></h3>

<p><font color= ”46BB0A”>Expected results :</font></p>
<p>Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.<br/>
A bacterial lawn on the LB petri dishes without antibiotic.<br/>
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).</p>

<p><font color= ”46BB0A”>Obtained results:</font></p>
<p>We obtained expected results.</p>

                     <h3><font color =”94FAF1”>Interpretation</font></h3>

<p>The transformation worked. Colonies contain a plasmid with the Chloramphenicol/Ampicilline resistance gene, present in pSB1C3/pSB1A2. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria incorpore the correct plasmid.</p>

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                                <h4>iGEM IONIS</h4>
                                <p> We're a group of six different schools from the IONIS Education Group. For this
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