Difference between revisions of "Team:Ionis Paris/17 09 16"

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                         <div class="col-xs-12 col-sm-9">
 
                         <div class="col-xs-12 col-sm-9">
 
                             <div class="bloggrid_right">
 
                             <div class="bloggrid_right">
−
                                 <div class="blog_top">
+
                                  
−
                                    <h4 class="blog_topHd">
+
                                    <h2 class="blog_topHd"> <font color =”#279AD3”>PCR : on PA and PB</font></h2>  
−
                                    PCR : on PA and PB
+
                                   
−
                                    </h4>
+
−
                                  </div>        
+
−
                                 
+
  
−
                                    <h4 class="blog_topHd">Objectives</h4>                      
+
                                    <h3><font color =”94FAF1”> Objectives </font></h3>                    
 
             <p>The overall purpose is to create PA (Pr - RBS - XylR - Term) and PB (Pu - RBS - Gaussia - Term) from our biosensor.</p>
 
             <p>The overall purpose is to create PA (Pr - RBS - XylR - Term) and PB (Pu - RBS - Gaussia - Term) from our biosensor.</p>
  
−
                                     <h4 class="blog_topHd">Materials</h4>
+
                                     <h3><font color =”94FAF1”> Materials </font></h3>
 
<p>DNA fragments :  BB123mut-6 (from mini prep 07/09)<br/>
 
<p>DNA fragments :  BB123mut-6 (from mini prep 07/09)<br/>
 
Primers: A12 (forward) and BBA-R (reverse) for PA, and BBB-F (forward) and A13 (reverse) for PB.</p>
 
Primers: A12 (forward) and BBA-R (reverse) for PA, and BBB-F (forward) and A13 (reverse) for PB.</p>
  
−
                                    <h4 class="blog_topHd">Protocol</h4>  
+
                                <h3><font color =”94FAF1”> Protocol </font></h3>
 
                                      
 
                                      
−
                                          <h3>PCR</h3>
+
                                      <h5><font color =”#3CB5E1”>PCR</font></h5>  
  
 
<ol>
 
<ol>
Line 65: Line 57:
 
         <li><p>2 µL of DNA fragment (BB123mut) or 2 µL H20 (Controls A and B)</p></li>
 
         <li><p>2 µL of DNA fragment (BB123mut) or 2 µL H20 (Controls A and B)</p></li>
 
     </ul></li>
 
     </ul></li>
−
</ol>
+
<li><p>—> Gently mix the reaction and perform a short spin centrifugation</p></li>
−
<p>
+
−
—> Gently mix the reaction and perform a short spin centrifugation</p>
+
  
 +
<li><p>Set the following parameters for the PCR reaction :</p>
 +
       
 +
        <ul><li><p><u>PA (2285 bp)</u></p></li>
 +
              <li><p>Lid temperature: 95°C</p></li>
 +
              <li><p>Initial denaturation : 95°C, 30s</p></li>
 +
              <li><p>30 cycles of :</p>
 +
                      <ul><li><p>95°C, 30 s</p></li>
 +
                          <li><p>58°C, 60 s</p></li>
 +
                          <li><p>68°C, 2 min 17 s</p></li>
 +
                      </ul></li>
 +
              <li><p>Final extension : 68°C, 5 min</li></p>
 +
              <li><p>Hold : 4°C</li></p>
 +
        </ul>
  
−
<li><p>50 µL Mix</p></li>
+
        <ul><li><p><u>PB (1037 bp)</u></p></li>
−
<li><p>One colony from the different petri dishes (pick one colony, put some on a new plate, divided into squares, and put the remaining bacteria into the PCR mix)</p></li>
+
              <li><p>Lid temperature: 95°C</p></li>
 +
              <li><p>Initial denaturation : 95°C, 30s</p></li>
 +
              <li><p>30 cycles of :</p>
 +
                      <ul><li><p>95°C, 30 s</p></li>
 +
                          <li><p>58°C, 60 s</p></li>
 +
                          <li><p>68°C, 1 min 02 s</p></li>
 +
                      </ul></li>
 +
              <li><p>Final extension : 68°C, 5 min</li></p>
 +
              <li><p>Hold : 4°C</li></p>
 +
        </ul>
  
−
                                <figure class="postImg">
+
        </ul></li>
−
                                    <img src="https://static.igem.org/mediawiki/2016/c/c8/T--Ionis_Paris--Notebook06_09Fig.1.png" alt="">
+
−
                                </figure>
+
  
−
<p>Gently mix the reaction</p>
 
  
−
<p>2.  Short spin centrifugation</p>
 
−
<p>3  Set the following parameters for the PCR reaction :</p>
 
  
−
          <li><p>P12 (2.195 kb)<br/>
 
−
              Lid température 95°C<br/>
 
−
              Initial denaturation : 95°C, 5 min<br/>
 
−
              30 cycles of : 95°C, 30 s<br/>
 
−
                                  58°C, 60 s<br/>
 
−
                                  68°C, 2 min 12 s<br/>
 
−
              Final extension : 68°C, 5 min<br/>
 
−
              Hold : 4°C</p></li>
 
−
          <li><p>P123 (3.4 kb)<br/>
 
−
              Lid température 95°C<br/>
 
−
              Initial denaturation : 95°C, 5 min<br/>
 
−
              30 cycles of : 95°C, 30 s<br/>
 
−
                                  58°C, 60 s<br/>
 
−
                                  68°C, 3 min 24 s<br/>
 
−
              Final extension : 68°C, 5 min<br/>
 
−
              Hold : 4°C</p></li>
 
  
−
<h3>Electrophoresis: for screening the PCR results</h3>
+
 
 +
<h5><font color =”#3CB5E1”>Electrophoresis: for screening the PCR results</font></h5>  
  
 
<p>1% Agarose gel:</p>
 
<p>1% Agarose gel:</p>
 
<ol>
 
<ol>
 
<li><p>Put 1 g of agarose + 100 mL of TAE 1X in a bottle of 500 mL</p></li>
 
<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>Mix and heat it 2 min 30 s in the microwave. 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>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>Flow the gel and place the combs</p></li>
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                                 <figure class="postImg">
 
                                 <figure class="postImg">
−
                                     <img src="https://static.igem.org/mediawiki/2016/b/b9/T--Ionis_Paris--Notebook06_09Fig.2.png" alt="">
+
                                     <img src="https://static.igem.org/mediawiki/2016/1/1e/T--Ionis_Paris--Notebook17_09Table1.png" alt="">
 
                                 </figure>
 
                                 </figure>
  
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</ol>
 
</ol>
  
−
<h3>Mini-culture: bacteria transformed with BB12mut and BB123mut</h3>
+
<h5><font color =”#3CB5E1”>PCR Purification</font></h5>  
−
<p>16 Mini-cultures of bacteria transformed with BB12mut (3, 4, 5, 7, 8, 9, 11, 12) and BB123mut (1, 2, 4, 5, 7, 10, 11, 13).<br/>
+
<p>QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available on <a href="https://www.qiagen.com/fi/resources/resourcedetail?id=390a728a-e6fc-43f7-bf59-b12091cc4380&lang=en"><font colo ="Deep Pink">this link</font></a>).</p>
−
Put the colony with satisfying PCR results from the plates divided into squares to a 50mL Falcon tube containing 5mL LB+Cm.</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 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>Calculate the quantity of DNA with the Nanodrop.</p></li>
 +
    <li><p>Store the purified DNA at -20°C.</p></li>
 +
</ol>
 
    
 
    
−
                                    <h4 class="blog_topHd">Results</h4>  
+
                                  <h3><font color =”94FAF1”> Results </font></h3>
 
                                      
 
                                      
−
<p>1st electrophoresis: Expected results / Obtained results </p>
+
<p><font color= ”46BB0A”> Expected results / Obtained results:</font></p>
  
−
                                <figure class="postImg">
 
−
                                    <img src="https://static.igem.org/mediawiki/2016/a/a1/T--Ionis_Paris--Notebook06_09Fig.3.png" alt="">
 
−
                                </figure>
 
  
 +
                                  <div class="col-md-6">
 +
                                                    <figure>
 +
                                                        <img src="https://static.igem.org/mediawiki/2016/2/2d/T--Ionis_Paris--Notebook17_09Fig2.png" alt="">
 +
                                                    </figure>
 +
                                            </div>
  
−
<p>2nd electrophoresis: Expected results / Obtained results</p>
 
  
−
                                <figure class="postImg">
+
                                  <div class="col-md-6">
−
                                    <img src="https://static.igem.org/mediawiki/2016/f/f3/T--Ionis_Paris--Notebook06_09Fig.4.png" alt="">
+
                                                    <figure>
−
                                </figure>                            
+
                                                        <img src="https://static.igem.org/mediawiki/2016/9/9b/T--Ionis_Paris--Notebook17_09Fig1.png" alt="">
 +
                                                    </figure>  
 +
                                            </div>
  
−
                                        <h4 class="blog_topHd">Interpretation</h4>
 
−
<p>We obtain the desired strip for BB12mut, for all the colonies (n°1 to 14). As shown on the gel above, the strips are closed to 2.2 kb, which is the size of P1+P2. A sequencing is necessary to be sure of the obtained biobrick.</p>
 
  
−
<p>We obtain the desired strip for BB123, for all the colonies except for n°7. As shown on the gel above, the strips are closed to 3.4 kb, which is the size of P1+P2+P3. A sequencing is necessary to be sure of the obtained biobrick.</p>
+
 
 +
                                  </div>
 +
                                  <div class="col-md-11">
 +
 
 +
                                        <h3><font color =”94FAF1”> Interpretation</font></h3>
 +
<p>
 +
We obtain the desired strip for PA and PB, as shown on the gel above the strips are closed to 2,285 bp and 1,037 bp.<br/>
 +
It seems that PA and PB have been properly amplified.</p>
 +
 
  
 
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Latest revision as of 01:25, 20 October 2016

PCR : on PA and PB

Objectives

The overall purpose is to create PA (Pr - RBS - XylR - Term) and PB (Pu - RBS - Gaussia - Term) from our biosensor.

Materials

DNA fragments : BB123mut-6 (from mini prep 07/09)
Primers: A12 (forward) and BBA-R (reverse) for PA, and BBB-F (forward) and A13 (reverse) for PB.

Protocol

PCR

  1. Mix for 5 samples (Total volume of Mix: 230 µL), in an Eppendorf tube:

    • 198.75 µL H2O

    • 25 µL Buffer Taq (1 X final, NEB #B9014S)

    • 5 µL dNTP (200 µM final, NEB #N0447S)

    • 1.25 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S)

  2. Add in 4 PCR tubes, in the following order:

    • 46 µL Mix

    • 1 µL primer forward (A12 or BBB-F)

    • 1 µL primer reverse (BBA-R or A13)

    • 2 µL of DNA fragment (BB123mut) or 2 µL H20 (Controls A and B)

  3. —> Gently mix the reaction and perform a short spin centrifugation

  4. Set the following parameters for the PCR reaction :

    • PA (2285 bp)

    • Lid temperature: 95°C

    • Initial denaturation : 95°C, 30s

    • 30 cycles of :

      • 95°C, 30 s

      • 58°C, 60 s

      • 68°C, 2 min 17 s

    • Final extension : 68°C, 5 min

    • Hold : 4°C

    • PB (1037 bp)

    • Lid temperature: 95°C

    • Initial denaturation : 95°C, 30s

    • 30 cycles of :

      • 95°C, 30 s

      • 58°C, 60 s

      • 68°C, 1 min 02 s

    • Final extension : 68°C, 5 min

    • Hold : 4°C

    5. Electrophoresis: for screening the PCR results

    1% Agarose gel:

    1. Put 1 g of agarose + 100 mL of TAE 1X in a bottle of 500 mL

    2. Mix and heat it 2 min 30 s in the microwave. Wait the cooling of the bottle until it is tepid.

    3. Add 5 µL of Gel Red 10,000 X (0.5 X final)

    4. Flow the gel and place the combs

    5. Wait until it is solidified. Remove slowly the combs.

    Drop-off:

    1. Short Speed centrifugation of samples

    2. Addition of 2 µL of Purple loading dye 6 X in 10 µL of sample

    3. Drop-off 10 µL of Purple ladder and 12 µL of each samples.

    4. Run at 90 V.

    PCR Purification

    QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available on this link).

    1. 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.

    2. Load the sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through

    3. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through.

    4. Centrifuge once more for 1 min at 13,000 rpm.

    5. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.

    6. 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.

    7. Calculate the quantity of DNA with the Nanodrop.

    8. Store the purified DNA at -20°C.

    Results

    Expected results / Obtained results:

Interpretation

We obtain the desired strip for PA and PB, as shown on the gel above the strips are closed to 2,285 bp and 1,037 bp.
It seems that PA and PB have been properly amplified.

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