Difference between revisions of "Team:Ionis Paris/20 09 16"

(Undo revision 426032 by ThomasJaisser (talk))
Line 1: Line 1:
 
{{IONIS_HEADER}}   
 
{{IONIS_HEADER}}   
 
<html>  
 
<html>  
−
     
 
  
 
     <!-- Nos feuilles de style -->
 
     <!-- Nos feuilles de style -->
Line 15: Line 14:
 
                     <div class="col-sm-12">
 
                     <div class="col-sm-12">
 
                         <div class="banner_title">
 
                         <div class="banner_title">
−
                             <h1 id="back_to_the_top">September 19th 2016</h1>
+
                             <h1 id="back_to_the_top">September 20th 2016</h1>
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
Line 30: Line 29:
 
                         <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">
−
                                    <h2 class="blog_topHd"> <font color =”#279AD3”>Digestion: PA and PB</font></h2>  
+
                                    <h2 class="blog_topHd">
−
                                         
+
                                    Digestion: PA and PB
 +
                                    </h2>
 +
                                  </div>        
 
                                    
 
                                    
−
                                  <h3><font color =”94FAF1”> Objectives </font></h3>                   
+
                                    <h4 class="blog_topHd">Objectives</h4>                      
−
             <p>Double digestion of PA, PB and pSB1C3-RFP by EcoRI and PstI for the subsequent ligation of PA and PB in pSB1C3.</p>
+
             <p>
 +
    Transformation: competent DH5⍺ cells with ligation products BBA and BBB
 +
</p>
  
−
                                  <h3><font color =”94FAF1”> Materials </font></h3>
 
  
−
                                      <h5><font color =”#3CB5E1”>Stock concentrations</font></h5>  
+
                                        <h2 class="blog_topHd">Transformation: competent DH5⍺ cells with ligation product BBA and BBB</h2>
  
−
<p>PA: ~20 ng/µL (from PCR purification 17/09)<br/>
+
                                    <h4 class="blog_topHd">Objectives</h4>                      
−
PB: ~20 ng/µL (from PCR purification 17/09)<br/>
+
            <p>The objective is to transform competent DH5⍺ cells with the ligations products BBA and BBB.</p>
−
pSB1C3-RFP 3: 104.14 ng/µL (from mini prep 19/07)</p>
+
  
−
                                  <h5><font color =”#3CB5E1”>Quantity of DNA required for the ligation of PA and PB into pSB1C3:</font></h5>  
+
                                    <h4 class="blog_topHd">Materials</h4>
  
−
<p>PA: Digestion of 75 ng (ratio 1:1 = 55.19 ng of digested PA needed)<br/>
 
−
PB: Digestion of 75 ng (ratio 2:1 = 50.10 ng of digested PB needed)<br/>
 
−
pSB1C3-RFP: Digestion of 200 ng (50 ng per part needed —> 100 ng of digested pSB1C3 needed —> 152 ng needed)</p>
 
  
  
−
                                  <h3><font color =”94FAF1”> Protocol </font></h3>
+
<ul><li>2 aliquots of "old" DH5⍺ Competent cells (from the 23/07/16)</li>
−
                                   
+
<li>2 aliquots of "new" DH5⍺ Competent cells (from the 20/09/16)</li>
−
                                      <h5><font color =”#3CB5E1”>Digestion</font></h5>  
+
<li>Plasmid DNA : Ligation product BBA and BBB (from the 19/09/16)</li>
 +
<li>Petri dish LB+Cm: Cm concentration = 25 µg/mL</li></ul>
  
  
−
<p>In a 1.5 mL Eppendorf tube, adding in the respected order (bigger volume first and enzyme last) :</p>
+
                                    <h4 class="blog_topHd">Protocol</h4>   
 +
                                   
 +
<h3>Experimental conditions realized :</h3>
  
  
 
                                 <figure class="postImg">
 
                                 <figure class="postImg">
−
                                     <img src="https://static.igem.org/mediawiki/2016/9/99/T--Ionis_Paris--Notebook18_09Table1.png" alt="">
+
                                     <img src="https://static.igem.org/mediawiki/2016/6/6b/T--Ionis_Paris--Notebook20_09Table1.png" alt="">
 
                                 </figure>
 
                                 </figure>
  
−
<ul><li>Short Spin Centrifugation</li>
 
−
    <li>Incubation 1h at 37°C</li>
 
−
    <li>Store at 4°C before gel electrophoresis and purification</li></ul>
 
−
 
−
                                  <h5><font color =”#3CB5E1”>Electrophoresis for digested pSB1C3-RFP :</font></h5>
 
−
 
−
<p>1% Agarose gel:</p>
 
  
 +
<h3>Transformation protocol:</h3>
 
<ol>
 
<ol>
−
     <li>Put 1 g of agarose low melting point + 100 mL of TAE 1X in a bottle of 500 mL.</li>
+
     <li>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.</li>
−
     <li>Mix and heat it 2min 30s in the microwaves. Wait the cooling of the bottle until it is tepid.</li>
+
     <li>Add the 10 µL plasmid DNA to the cell mixture.</li>
−
     <li>Add 5 µL of Gel Red 10,000X (0.5 X final).</li>
+
    <li>Carefully flick the tubes 4-5 times to mix cells and DNA. <b>Do not vortex.</b></li>
−
     <li>Flow the gel and place the combs.</li>
+
     <li>Place on ice for 30 min. Do not mix.</li>
−
     <li>Wait until it is solidified. Remove slowly the combs.</li>
+
    <li>Heat shock at exactly 42°C for 45 s. Do not mix.</li>
 +
    <li>Place on ice for 5 min. Do not mix.</li>
 +
    <li>Pipette 250 µL of room temperature SOC into the mixture.</li>
 +
    <li>Place at 37°C for 1h at 250 rpm.</li>
 +
     <li>Warm selection plates to 25°C.</li>
 +
    <li>Mix the cells thoroughly by flicking the tubes and inverting.</li>
 +
     <li>Spread the corresponding volume onto each plate.</li>
 +
    <li>Incubate all the plates O/N at 37°C.</li>
 
</ol>
 
</ol>
  
 +
                                    <h4 class="blog_topHd">Results (obtained on the 21/09)</h4>   
  
−
<p>Drop-off:</p>
+
<p><b>Expected Results</b></p>
  
−
<ol>
+
<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/>
−
    <li>Short Speed centrifugation of samples.</li>
+
More colonies on the petri dishes plated with the « new » competent bacteria (from 20/09) transformed with the different ligation products.<br/>
−
    <li>Addition of 4 µL of Purple loading dye 6X in the 20 µL of each samples.</li>
+
A bacterial lawn on the LB petri dishes without antibiotic.<br/>
−
    <li>Drop-off 10 µL of Purple ladder and 24 µL of each samples.</li>
+
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).</p>
−
</ol>
+
−
<p>Plan:</p>
+
  
  
 +
<p><b>Obtained Results</b></p>
 +
<p>We obtained expected results.
  
−
                                <figure class="postImg">
+
</p>
−
                                    <img src="https://static.igem.org/mediawiki/2016/a/ad/T--Ionis_Paris--Notebook18_09Table2.png" alt="">
+
−
                                </figure>
+
  
 +
                                    <h4 class="blog_topHd">Interpretation</h4> 
  
−
<p>Run at 100V</p>
 
  
 +
<p>The transformation worked. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria integrated the correct plasmid.</p>
  
−
  <h5><font color =”#3CB5E1”>Gel purification for digested pSB1C3:</font></h5>
 
−
<p>QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier (available on <a href="https://www.qiagen.com/us/resources/resourcedetail?id=f4ba2d24-8218-452c-ad6f-1b6f43194425&lang=en"><font color = "DeepPink">this link</font></a>).</p>
 
−
<ol>
 
−
    <li>Excise the DNA fragment from the agarose gel. Gel slice Weigh = 0.250 g</li>
 
−
    <li>Add 3 volumes Buffer QG (750 µL) to 1 volume of gel.</li>
 
−
    <li>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.</li>
 
−
    <li>Add 1 gel volume isopropanol to the sample and mix.</li>
 
−
    <li>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.</li>
 
−
    <li>Add 500 µL Buffer QG. Centrifuge for 1 min at 13,000 rpm and discard flow-through.</li>
 
−
    <li>Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through.</li>
 
−
    <li>Centrifuge once more for 1 min at 13,000 rpm.</li>
 
−
    <li>Place QIAquick column into a clean 1.5 mL microcentrifuge tube.</li>
 
−
    <li>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.</li>
 
−
    <li>Store the purified DNA at 4°C before the ligation.</li>
 
  
−
</ol>
+
                                        <h2 class="blog_topHd">Competent cells: E.Coli DH5⍺</h2>
  
  
 +
                                    <h4 class="blog_topHd">Objectives</h4>                     
 +
            <p>The objective is to make a stock of E.Coli DH5⍺ competent cells for subsequent transformations.</p>
  
 +
                                    <h4 class="blog_topHd">Materials</h4>
  
  
−
<ol>
 
−
<li><p>Mix for 5 samples (Total volume of Mix: 230 µL), in an Eppendorf tube:</p>
 
−
    <ul><li><p>198.75 µL H2O</p></li>
 
−
        <li><p>25 µL Buffer Taq (1 X final, NEB #B9014S)</p></li>
 
−
        <li><p>5 µL dNTP (200 µM final, NEB #N0447S)</p></li>
 
−
        <li><p>1.25 µL Taq polymerase (2.5 units / 50 µL PCR final, NEB #M0273S)</p></li>
 
−
    </ul></li>
 
−
<li><p>Add in 4 PCR tubes, in the following order:</p>
 
−
    <ul><li><p>46 µL Mix</p></li>
 
−
        <li><p>1 µL primer forward (A12 or BBB-F)</p></li>
 
−
        <li><p>1 µL primer reverse (BBA-R or A13)</p></li>
 
−
        <li><p>2 µL of DNA fragment (BB123mut) or 2 µL H20 (Controls A and B)</p></li>
 
−
    </ul></li>
 
−
<li><p>—> Gently mix the reaction and perform a short spin centrifugation</p></li>
 
  
−
<li><p>Set the following parameters for the PCR reaction :</p>
+
<ul><li>O/N DH5⍺ pre-culture (made the 22/07): O/N inoculation of 100 µL DH5⍺ into 100 mL LB.</li>
−
       
+
<li>0.1M CaCl2: prepared the 23/07</li>
−
        <ul><li><p><u>PA (2285 bp)</u></p></li>
+
<li>0.1M CaCl2/15% Glycerol: prepared on the 23/07</li></ul>
−
              <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>
+
  
−
        <ul><li><p><u>PB (1037 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, 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>
 
  
−
        </ul></li>
+
                                    <h4 class="blog_topHd">Protocol</h4>   
 +
                                   
 +
<h3>Competence</h3>
  
−
 
−
 
−
 
−
 
−
<h5><font color =”#3CB5E1”>Electrophoresis: for screening the PCR results</font></h5>
 
−
 
−
<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>Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer.</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>Incubation at 250 rpm at 37°C until the DO 0,6 —> DO = 0.591</li>
−
<li><p>Add 5 µL of Gel Red 10,000 X (0.5 X final)</p></li>
+
    <li>Cells were transferred to 3 sterile ice-cold 50 mL Falcon tubes. 20 mL in each falcon tube.</li>
−
<li><p>Flow the gel and place the combs</p></li>
+
    <li>Incubate on ice for 30 min. Do not allow cells to warm up over 4°C.</li>
−
<li><p>Wait until it is solidified. Remove slowly the combs.</p></li>
+
    <li>Spin cells at 4000 rpm for 10 min at 4°C.</li>
 +
    <li>Discard supernatant and try to drain all remaining media.</li>
 +
    <li>Gently resuspend on 10 mL cold 0.1M CaCl2</li>
 +
    <li>Incubate on ice for 20 min</li>
 +
    <li>Centrifuge 10 min at 4,000 rpm at 4°C</li>
 +
    <li>Discard supernatant and gently resuspend on 5 mL cold 0.1M CaCl2/25% Glycerol</li>
 +
    <li>Transfer in 1.5 mL eppendorf (100 µL/tube)</li>
 +
    <li>Store at -80°C</li>
 
</ol>
 
</ol>
  
−
<p>Drop-off:</p>
+
<p>NB: The competency of the prepared cells will be tested on the 20/09.</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 samples.</p></li>
+
−
 
+
−
                                <figure class="postImg">
+
−
                                    <img src="https://static.igem.org/mediawiki/2016/1/1e/T--Ionis_Paris--Notebook17_09Table1.png" alt="">
+
−
                                </figure>
+
−
 
+
−
<li><p>Run at 90 V.</p></li>
+
−
 
+
−
 
+
−
                            <h3><font color =”94FAF1”> Results </font></h3>
+
−
 
+
−
<h5><font color =”#3CB5E1”>Electrophoresis</font></h5>
+
−
                                   
+
−
<p><font color= ”46BB0A”> Expected results / Obtained results:</font></p>
+
−
 
+
−
 
+
−
                                  <div class="col-md-6">
+
−
                                                    <figure>
+
−
                                                        <img src="https://static.igem.org/mediawiki/2016/c/c9/T--Ionis_Paris--Notebook18_09Fig1.png" alt="">
+
−
                                                    </figure>
+
−
                                            </div>
+
−
 
+
−
 
+
−
                                  <div class="col-md-6">
+
−
                                                    <figure>
+
−
                                                        <img src="https://static.igem.org/mediawiki/2016/2/29/T--Ionis_Paris--Notebook19_09Fig2.png" alt="">
+
−
                                                    </figure>
+
−
                                            </div>
+
−
 
+
−
 
+
−
 
+
−
                                  </div>
+
−
                                  <div class="col-md-11">
+
−
 
+
−
                                      <h2 class="blog_topHd"> <font color =”#279AD3”>Ligation of PA and PB in pSB1C3 :</font></h2>
+
−
 
+
−
 
+
−
                                    <h3><font color =”94FAF1”> Objectives </font></h3>                     
+
−
            <p>Ligation of PA and PB in pSB1C3 for subsequent transformation and creation of a stock a bacteria containing the two parts of our biosensor.<br/>
+
−
The molar ratios for the ligations were calculated using <a href="http://nebiocalculator.neb.com/#!/ligation"><font color = "Deepink">NEB BioCalculator</font></a></p>
+
−
 
+
−
                                  <h3><font color =”94FAF1”> Materials </font></h3>
+
−
 
+
−
                              <h5><font color =”#3CB5E1”>Concentrations of the different components after digesion and PCR or gel purification :</font></h5>
+
−
 
+
−
<p>pSB1C3 : 4.33 ng/µL (130 ng / 30 µL)<br/>
+
−
PA : 2.5 ng/µL (75 ng / 30 µL)<br/>
+
−
PB : 2.5 ng/µL (75 ng / 30 µL)</p>
+
−
 
+
−
 
+
−
                              <h3><font color =”94FAF1”> Protocol </font></h3>
+
−
                                   
+
−
<p>In the following order, add:</p>
+
−
 
+
−
 
+
−
                                <figure class="postImg">
+
−
                                    <img src="https://static.igem.org/mediawiki/2016/1/15/T--Ionis_Paris--Notebook18_09Table3.png" alt="">
+
−
                                </figure>
+
  
−
<p>Mix by pipetting</p>
 
−
<p>Incubate 1h at Room Temperature</p>
 
  
  
Line 314: Line 209:
 
         <!-- ====END BLOG TABLE==== -->
 
         <!-- ====END BLOG TABLE==== -->
 
          
 
          
−
       
+
  <!-- ====START SOCIAL Link==== -->
−
    <!-- ====START SOCIAL Link==== -->
+
 
     <div class="footer_social">
 
     <div class="footer_social">
 
         <div class="container-fluid">
 
         <div class="container-fluid">

Revision as of 02:16, 20 October 2016

Digestion: PA and PB

Objectives

Transformation: competent DH5⍺ cells with ligation products BBA and BBB

Transformation: competent DH5⍺ cells with ligation product BBA and BBB

Objectives

The objective is to transform competent DH5⍺ cells with the ligations products BBA and BBB.

Materials

  • 2 aliquots of "old" DH5⍺ Competent cells (from the 23/07/16)
  • 2 aliquots of "new" DH5⍺ Competent cells (from the 20/09/16)
  • Plasmid DNA : Ligation product BBA and BBB (from the 19/09/16)
  • Petri dish LB+Cm: Cm concentration = 25 µg/mL

Protocol

Experimental conditions realized :

Transformation protocol:

  1. 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.
  2. Add the 10 µL plasmid DNA to the cell mixture.
  3. Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex.
  4. Place on ice for 30 min. Do not mix.
  5. Heat shock at exactly 42°C for 45 s. Do not mix.
  6. Place on ice for 5 min. Do not mix.
  7. Pipette 250 µL of room temperature SOC into the mixture.
  8. Place at 37°C for 1h at 250 rpm.
  9. Warm selection plates to 25°C.
  10. Mix the cells thoroughly by flicking the tubes and inverting.
  11. Spread the corresponding volume onto each plate.
  12. Incubate all the plates O/N at 37°C.

Results (obtained on the 21/09)

Expected Results

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.
More colonies on the petri dishes plated with the « new » competent bacteria (from 20/09) transformed with the different ligation products.
A bacterial lawn on the LB petri dishes without antibiotic.
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).

Obtained Results

We obtained expected results.

Interpretation

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

Competent cells: E.Coli DH5⍺

Objectives

The objective is to make a stock of E.Coli DH5⍺ competent cells for subsequent transformations.

Materials

  • O/N DH5⍺ pre-culture (made the 22/07): O/N inoculation of 100 µL DH5⍺ into 100 mL LB.
  • 0.1M CaCl2: prepared the 23/07
  • 0.1M CaCl2/15% Glycerol: prepared on the 23/07

Protocol

Competence

  1. Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer.
  2. Incubation at 250 rpm at 37°C until the DO 0,6 —> DO = 0.591
  3. Cells were transferred to 3 sterile ice-cold 50 mL Falcon tubes. 20 mL in each falcon tube.
  4. Incubate on ice for 30 min. Do not allow cells to warm up over 4°C.
  5. Spin cells at 4000 rpm for 10 min at 4°C.
  6. Discard supernatant and try to drain all remaining media.
  7. Gently resuspend on 10 mL cold 0.1M CaCl2
  8. Incubate on ice for 20 min
  9. Centrifuge 10 min at 4,000 rpm at 4°C
  10. Discard supernatant and gently resuspend on 5 mL cold 0.1M CaCl2/25% Glycerol
  11. Transfer in 1.5 mL eppendorf (100 µL/tube)
  12. Store at -80°C

NB: The competency of the prepared cells will be tested on the 20/09.

Creative and impressive building

How you can be a super creative

World best photographer and photography