Difference between revisions of "Team:Valencia UPV/Notebook"

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<body>
 
<body>
  
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18/05
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<section>
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Take glycerinated cultures of C58 Agrobacterium with dsRED from GoldenBraid Collection. Prepare broth culture (5 mL LB + 5 uL kanamycin + 5 uL Rifampin) 1:1000 and incubate overnight at 28^C.  
+
<div class="container">
−
19/05
+
<div class="timeline">
−
Refresh previously made culture by inoculating 10 uL in a new culture medium.
+
<div class="timeline-hline"></div>
−
20/05
+
<div class="blog-post-item">
−
|Agroinfiltration path:#; in Nicotiana benthamiana of C58 with dsRED.
+
  <div class="timeline-entry rounded">
−
06/06
+
      18<span>May</span>
−
<l>Take from the glycerinates of Goldenbraid Collection:</l>
+
      <div class="timeline-vline"></div>
−
<t>     
+
  </div>
−
   Plasmid GB Code
+
  <ul class="blog-post-info list-inline">
−
  pD6B3 alpha1 GB0015
+
      <li><span class="font-lato">May 18, 2016</span></li>
−
  pD6B3 alpha2 GB0017
+
  </ul>
−
  pD6B3 omega1 GB0019
+
  <p><a href=" glycerinated cultures of C58 <i>Agrobacterium</i> with dsRED from GoldenBraid Collection. Prepare broth culture (5 mL LB + 5 &mu"></a>L kanamycin + 5 μL Rifampin) 1:1000 and incubate overnight at 28^C. <br></p>
−
  pD6B3 omega2 GB0021
+
</div>
−
  pUPD2 GB0307
+
<div class="blog-post-item">
−
</t>
+
  <div class="timeline-entry rounded">
−
Prepare liquid culture (3 mL LB + 3 uL antibiotic) 1:1000. Incubate at 37^C overnight.  
+
      19<span>May</span>
−
<l>Experiment with snails:</l>
+
      <div class="timeline-vline"></div>
−
Two experiments: one with infiltrated Nicotiana benthamiana and another with not infiltrated N.benthamiana (negative control). Lettuce leafs haven't been correctly infiltrated and it seems that the expression level is low.
+
  </div>
−
Let both N. benthamiana leafs with snails overnight at room temperature in separated boxes. We will observe if snails eat the leafs and if appears fluorescence.  
+
  <ul class="blog-post-info list-inline">
−
15/06
+
      <li><span class="font-lato">May 19, 2016</span></li>
−
Experiment is over due to snails haven't eaten leafs enough so we have not been able to see fluorescence.
+
  </ul>
−
30/06
+
  <p><a href="esh previously made culture by inoculating 10 &mu"></a>L in a new culture medium.<br></p>
−
Orange DNA Genome Extraction protocol href
+
</div>
−
<l>Take from the glycerinates of GoldenBraid Collection:</l>
+
<div class="blog-post-item">
−
<t>
+
  <div class="timeline-entry rounded">
−
   Plasmid GB Code
+
      20<span>May</span>
−
  Promoter 35s:Cas9:nopaline synthase terminator (Tnos) GB0639
+
      <div class="timeline-vline"></div>
−
  Luciferase (Luc) in pUPD2 GB0096
+
  </div>
−
  Tnos in pUPD2 GB0037
+
  <ul class="blog-post-info list-inline">
−
</t>
+
      <li><span class="font-lato">May 20, 2016</span></li>
−
01/07
+
  </ul>
−
<l>
+
  <p><a href="#">Agroinfiltration</a> in <i>Nicotiana benthamiana</i> of C58 with dsRED.<br></p>
−
Miniprep with E.Z.N.A.  Plasmid Mini Kit I, Q(capless) Spin of:  
+
</div>
−
<l>Promoter 35s:Cas9 : Tnos</l>
+
<div class="blog-post-item">
−
<l>Luciferase and nopaline synthase terminator cultures haven't succeed. Repeat Luc and Tnos cultures. </l>
+
  <div class="timeline-entry rounded">
−
Primers IG16JUN01 and IG16JUN02 have arrived.
+
      06<span>Jun</span>
−
Finish orange DNA Genome Extraction and check DNA concentration with NanoDrop. Concentration is very low so extraction will be done again.
+
      <div class="timeline-vline"></div>
−
02/07
+
  </div>
−
<l>
+
  <ul class="blog-post-info list-inline">
−
Miniprep with E.Z.N.A.  Plasmid Mini Kit I, Q(capless) Spin of:
+
      <li><span class="font-lato">June 06, 2016</span></li>
−
<l>
+
  </ul>
−
Luc in pUPD2
+
  <p></p>
−
<l>Tnos in pUPD2</l>
+
  <ul>
−
<l>Check orange DNA genome concentration with NanoDrop. </l>
+
      <li>
−
<t>
+
        href="
−
   Sample DNA concentration (ng / uL)
+
        <l>
−
   Clemenules1 3153.8
+
        T"></a>
−
   Clemenules 2 4527.9
+
        <l>
−
</t>
+
        Take from the glycerinates of Goldenbraid Collection:
−
<l>Perform a PCR to bind linker with luciferase:</l>
+
      </li>
−
<t>
+
  </ul>
−
  Reagent Volume(uL) Program
+
   <p><a href=""></a><br></p>
−
  LuciferasepUPD 1 Temperature Time
+
   <div class="table-responsive" style="width:55%;overflow:inherit">
−
   Buffer HF 10 98^C 5 minutes
+
      <table class="table table-bordered table-striped">
−
   dNTPs 2 98^C 35x 30 seconds
+
        <tr>
−
   IG16JUN01 2.5 70^C 35x 30 seconds
+
            <td><a href="Plas"></a>Plasmid</td>
−
  IG16JUN02 2.5 72^C 35x 1 minute 30 seconds
+
            <td>GB Code</td>
−
  Taq phusion 0.5 72^C 10 minutes
+
        </tr>
−
  H2O milli-Q 31.5 16^C &infin;
+
        <tr>
−
</t>
+
            <td><a href="3 &alpha"></a>1</td>
−
04/07
+
            <td>GB0015</td>
−
Run electrophoresis gel of Clemenules DNA (agarose 1%). 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.
+
        </tr>
−
Rice DNA Genome Extraction Protocol href
+
        <tr>
−
Run electrophoresis gel of Luciferase PCR product. 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.
+
            <td><a href="3 &alpha"></a>2</td>
−
Ligate Reaction --> Linker:Luciferase into a pUPD2
+
            <td>GB0017</td>
−
Transform E.coli DH5alpha with it. The method that is necessary to carry out this procedure is explained in |protocols path:#;
+
        </tr>
−
Check DNA concentration with NanoDrop.
+
        <tr>
−
<t>
+
            <td><a href="3 &Omega"></a>1</td>
−
  SAMPLE DNA Concentration(ng / uL)
+
            <td>GB0019</td>
−
  Rice Gleva 1 22.8
+
        </tr>
−
  Rice Gleva 2 17.3
+
        <tr>
−
</t>
+
            <td><a href="3 &Omega"></a>2</td>
−
05/07
+
            <td>GB0021</td>
−
Run electrophoresis gel of Gleva rice DNA. We have checked that there isn't DNA.
+
        </tr>
−
Repeat: Rice DNA Genome Extraction href
+
        <tr>
−
Check DNA concentration with NanoDrop.
+
            <td><a href="pUPD"></a>pUPD2</td>
−
<t>
+
            <td>GB0307</td>
−
  SAMPLE DNA Concentration(ng / uL)
+
        </tr>
−
  Rice Gleva 1 294.9
+
      </table>
−
  Rice Gleva 2 193.7
+
  </div>
−
</t>
+
  <p><a href=""></a><br><a href="are liquid culture (3 mL LB + 3 &mu"></a>L antibiotic) 1:1000. Incubate at 37^C overnight. <br><a href=""></a><br></p>
−
Run electrophoresis gel of Gleva rice DNA. It observed that genome extraction is correctly done.
+
  <ul>
−
06/07
+
      <li>
−
Take glycerinated cultures from Goldenbraid Collection:
+
        href="
−
<t>
+
        <l>
−
   GB part Plasmid Antibiotic Number GB
+
        E"></a>
−
   psgRNA pUPD Ampicillin 0645
+
        <l>
−
   U6-26 pUPD Ampicillin 1001
+
        Experiment with snails:
−
</t>
+
      </li>
−
07/07
+
  </ul>
−
<l>
+
  <p><a href="experiments: one with infiltrated <i>Nicotiana benthamiana</i> and another with not infiltrated N.benthamiana (negative control). Lettuce leafs haven&rsquo"></a>t been correctly infiltrated and it seems that the expression level is low.<br><a href="Let "></a>Let both <i>N. benthamiana</i> leafs with snails overnight at room temperature in separated boxes. We will observe if snails eat the leafs and if appears fluorescence. <br><a href=""></a><br></p>
−
Miniprep with E.Z.N.A.  Plasmid Mini Kit I, Q(capless) Spin of:
+
</div>
−
<l>
+
<div class="blog-post-item">
−
psgRNA in pUPD2
+
  <div class="timeline-entry rounded">
−
<l>U6-26 in pUPD2</l>
+
      15<span>Jun</span>
−
Primers IG16JUL01, IG16JUL02, IG16JUL03, IG16JUL04, IG16JUL05, IG16JUL06, IG16JUL07 and IG16JUL08 arrived.
+
      <div class="timeline-vline"></div>
−
gBlocks of - promoter 35s:5' region - have arrived.
+
  </div>
−
We perform a PCR of orange and rice Genome Extraction following the |protocol path:#;
+
  <ul class="blog-post-info list-inline">
−
<t>
+
      <li><span class="font-lato">June 15, 2016</span></li>
−
  Sample Initial concentration(ng/uL) Final concentration(ng/uL) Initial volume(uL) Final volume (uL)
+
  </ul>
−
  Clemenules 1 3153.8 150 4.756 100
+
  <p><a href="riment is over due to snails haven&rsquo"></a>t eaten leafs enough so we have not been able to see fluorescence.<br></p>
−
  Clemenules 2 4527.9 150 3.31 100
+
</div>
−
  Gleva 1 294.9 150 50.8647 100
+
<div class="blog-post-item">
−
  Gleva 2 193.7 150 77.44 100
+
  <div class="timeline-entry rounded">
−
</t>
+
      30<span>Jun</span>
−
<t>
+
      <div class="timeline-vline"></div>
−
  REAGENT VOLUME(uL) PROGRAM
+
  </div>
−
  Clemenules DNA 1 TEMPERATURE TIME
+
  <ul class="blog-post-info list-inline">
−
  Buffer HF 10 98^C 5 minutes
+
      <li><span class="font-lato">June 30, 2016</span></li>
−
  dNTPs 2 98^C 35x 30 seconds
+
  </ul>
−
  IG16JUL01 (TFL_For) 2.5 64^C 35x 30 seconds
+
  <p><a href="Oran"></a>Orange DNA Genome Extraction protocol href<br><a href=""></a><br></p>
−
  IG16JUL02 (TFL_Rev) 2.5 72^C 35x 30 seconds
+
  <ul>
−
  Taq phusion 0.5 72^C 10 minutes
+
      <li>
−
  H2O milli-Q 31.5 16^C &infin;
+
        href="
−
</t>
+
        <l>
−
<t>
+
        T"></a>
−
   REAGENT VOLUME(uL) PROGRAM
+
        <l>
−
   Gleva DNA 1 TEMPERATURE TIME
+
        Take from the glycerinates of GoldenBraid Collection:
−
  Buffer HF 10 98^C 5 minutes
+
      </li>
−
  dNTPs 2 98^C 35x 30 seconds
+
  </ul>
−
  IG16JUL03 (Ga20_for) 2.5 72^C 35x 30 seconds
+
   <p><a href=""></a><br></p>
−
   IG16JUL02 (Ga20_rev) 2.5 72^C 35x 30 seconds
+
  <div class="table-responsive" style="width:55%;overflow:inherit">
−
   Taq phusion 0.5 72^C 10 minutes
+
      <table class="table table-bordered table-striped">
−
  H2O milli-Q 31.5 16^C &infin;
+
        <tr>
−
</t>
+
            <td><a href="Plas"></a>Plasmid</td>
−
Ligate reaction of promoter 35s:5' region in pUPD2. Following ligation protocol href, BsmbI enzyme is used in this reaction.
+
            <td>GB Code</td>
−
08/07
+
        </tr>
−
Run electrophoresis gel of Clemenules and Gleva PCR products. 45 mL agarose gel at 1 % 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 120 V.
+
        <tr>
−
Transform E.coli with the next devise: promoter 35s:5' region (electroporation 2.5KV). Plating it and incubate overnight at 37^C.
+
            <th> href="Prom"></a>Promoter 35s:Cas9:nopaline synthase terminator (Tnos)</th>
−
Take glycerinated culture for Georgia collaboration. The devise is promoter 35s:GFP:Tnos (alpha1 and kanamycin)
+
            <th>GB0639</th>
−
09/07
+
        </tr>
−
<l>
+
        <tr>
−
Miniprep with E.Z.N.A.  Plasmid Mini Kit I, Q(capless) Spin of:
+
            <td><a href="Luci"></a>Luciferase (Luc) in pUPD2</td>
−
<l>promoter 35s:GFP:Tnos</l>
+
            <td>GB0096</td>
−
No colonies have grown in the devise promoter 35s:5' region petri dishes. Repeat transformation procedure, plating again and incubate overnight at 37^C.
+
        </tr>
−
11/07
+
        <tr>
−
Pick a single E. coli DH5alpha (promoter 35s:5' region in pUPD2) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 uL of chloramphenicol in a 50 ml tube with the colony and incubate it overnight at 37^C with shaking.
+
            <td><a href="Tnos"></a>Tnos in pUPD2</td>
−
Check Georgia miniprep concentration with NanoDrop (promoter 35s:GFP : Tnos)
+
            <td>GB0037</td>
−
<t>
+
        </tr>
−
  Sample DNA Concentration(ng / uL) DNA Concentration(ng)
+
      </table>
−
  1 105.2 5035.2
+
  </div>
−
  2 104.6 5035.2
+
  <p><a href=""></a><br></p>
−
</t>
+
</div>
−
Targets ligations in pUPD2 (Orange Clemenules and Rice Gleva). Following |ligation protocol path:#;, BsmbI enzyme is used in this reaction.
+
<div class="blog-post-item">
−
12/07
+
  <div class="timeline-entry rounded">
−
Pick a single E. coli DH5alpha (target Gleva in pUPD2 and target Clemenules in pUPD2) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 uL of chloramphenicol in a 50 ml tube with the colony and incubate it 16 hours at 37^C.
+
      01<span>Jul</span>
−
<l>
+
      <div class="timeline-vline"></div>
−
Miniprep with E.Z.N.A.  Plasmid Mini Kit I, Q(capless) Spin of:
+
  </div>
−
<l>Promoter 35s:5'region in pUPD2</l>
+
  <ul class="blog-post-info list-inline">
−
Digestion of minipreps with NotI. Incubate 1 hour at 37^C
+
      <li><span class="font-lato">July 01, 2016</span></li>
−
Run electrophoresis gel of the following devise: promoter 35s:5' region in pUPD2. We remain the samples 1 and 3.
+
  </ul>
−
<l>
+
  <p><a href="iniprep with E.Z.N.A ®"></a>.  Plasmid Mini Kit I, Q(capless) Spin of: <br></p>
−
Miniprep with E.Z.N.A.  Plasmid Mini Kit I, Q(capless) Spin of:
+
  <ul>
−
<l>
+
      <li>
−
Rice Gleva target in pUPD2
+
        href="
−
<l>Orange Clemenules target in pUPD2</l>
+
        <l>
−
Digestion of minipreps with NotI. Incubate 1 hour at 37^C.
+
        "></a>
−
<l>
+
        <l>
−
Run electrophoresis gel of the same devise:
+
        Promoter 35s:Cas9 : Tnos
−
<l>
+
      </li>
−
Rice Gleva target in pUPD2
+
  </ul>
−
<l>Orange Clemenules target in pUPD2</l>
+
  <p><a href=""></a><br><a href="uciferase and nopaline synthase terminator cultures haven&rsquo"></a>t succeed. Repeat Luc and Tnos cultures. <br>
−
<l>
+
  </ul>
−
Ligation using Golden Braid assembly of the next devise:
+
  <p><a href=""></a><br><a href="Prim"></a>Primers IG16JUN01 and IG16JUN02 have arrived.<br><a href=""></a><br><a href="Fini"></a>Finish orange DNA Genome Extraction and check DNA concentration with NanoDrop. Concentration is very low so extraction will be done again.<br><a href=""></a><br></p>
−
<l>Promoter 35s:5' region:Target:Luc:Tnos in alpha1 plasmid.</l>
+
</div>
 +
<div class="blog-post-item">
 +
  <div class="timeline-entry rounded">
 +
      02<span>Jul</span>
 +
      <div class="timeline-vline"></div>
 +
  </div>
 +
  <ul class="blog-post-info list-inline">
 +
      <li><span class="font-lato">July 02, 2016</span></li>
 +
  </ul>
 +
  <p><a href="iniprep with E.Z.N.A ®"></a>.  Plasmid Mini Kit I, Q(capless) Spin of:<br></p>
 +
  <ul>
 +
  <li>
 +
      href="
 +
      <l>
 +
      "></a>
 +
      <l>
 +
      Luc in pUPD2
 +
   </li>
 +
   </p>
 +
   <ul>
 +
      <li>
 +
        href="
 +
        <l>
 +
        "></a>
 +
        <l>
 +
        Tnos in pUPD2
 +
      </li>
 +
   </ul>
 +
   <p><a href=""></a><br></p>
 +
   <ul>
 +
      <li>
 +
        href="
 +
        <l>
 +
        C"></a>
 +
        <l>
 +
        Check orange DNA genome concentration with NanoDrop.  
 +
      </li>
 +
  </ul>
 +
  <p><a href=""></a><br></p>
 +
  <div class="table-responsive" style="width:55%;overflow:inherit">
 +
      <table class="table table-bordered table-striped">
 +
        <tr>
 +
            <td><a href="le</td><td>DNA concentration (ng / &mu"></a>L)</td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="Clem"></a>Clemenules1 </td>
 +
            <td>3153.8</td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="Clem"></a>Clemenules 2 </td>
 +
            <td>4527.9</td>
 +
        </tr>
 +
      </table>
 +
   </div>
 +
   <p><a href=""></a><br></p>
 +
   <ul>
 +
      <li>
 +
        href="
 +
        <l>
 +
        P"></a>
 +
        <l>
 +
        Perform a PCR to bind linker with luciferase:
 +
      </li>
 +
  </ul>
 +
  <p></p>
 +
  <div class="table-responsive" style="width:55%;overflow:inherit">
 +
      <table class="table table-bordered table-striped">
 +
        <tr>
 +
            <td><a href="ent</td><td>Volume(&mu"></a>L)</td>
 +
            <td>Program</td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="Luci"></a>LuciferasepUPD</td>
 +
            <td>1</td>
 +
            <td>Temperature</td>
 +
            <td>Time </td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="Buff"></a>Buffer HF</td>
 +
            <td>10</td>
 +
            <td>98^C</td>
 +
            <td>5 minutes</td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="dNTP"></a>dNTPs</td>
 +
            <td>2</td>
 +
            <td>98^C</td>
 +
            <td>35x</td>
 +
            <td>30 seconds</td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="IG16"></a>IG16JUN01</td>
 +
            <td>2.5</td>
 +
            <td>70^C</td>
 +
            <td>35x</td>
 +
            <td>30 seconds</td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="IG16"></a>IG16JUN02</td>
 +
            <td>2.5</td>
 +
            <td>72^C</td>
 +
            <td>35x</td>
 +
            <td>1 minute 30 seconds</td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="Taq "></a>Taq phusion</td>
 +
            <td>0.5</td>
 +
            <td>72^C</td>
 +
            <td>10 minutes</td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="b>2</sub>O milli-Q</td><td>31.5</td><td>16^C</td><td>&infin"></a></td>
 +
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   04<span>Jul</span>
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<ul class="blog-post-info list-inline">
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  <li><span class="font-lato">July 04, 2016</span></li>
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<p><a href=""></a><br><a href="Run "></a>Run electrophoresis gel of Clemenules DNA (agarose 1%). 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.<br><a href=""></a><br><a href="Rice"></a>Rice DNA Genome Extraction Protocol href<br><a href=""></a><br><a href="Run "></a>Run electrophoresis gel of Luciferase PCR product. 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.<br><a href=""></a><br><a href="Liga"></a>Ligate Reaction --> Linker:Luciferase into a pUPD2 <br><a href=""></a>
  
 
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Revision as of 22:33, 21 September 2016

18May
  • May 18, 2016

L kanamycin + 5 μL Rifampin) 1:1000 and incubate overnight at 28^C.

19May
  • May 19, 2016

L in a new culture medium.

20May
  • May 20, 2016

Agroinfiltration in Nicotiana benthamiana of C58 with dsRED.

06Jun
  • June 06, 2016

  • href=" T"> Take from the glycerinates of Goldenbraid Collection:


Plasmid GB Code
1 GB0015
2 GB0017
1 GB0019
2 GB0021
pUPD2 GB0307


L antibiotic) 1:1000. Incubate at 37^C overnight.

  • href=" E"> Experiment with snails:

t been correctly infiltrated and it seems that the expression level is low.
Let both N. benthamiana leafs with snails overnight at room temperature in separated boxes. We will observe if snails eat the leafs and if appears fluorescence.

15Jun
  • June 15, 2016

t eaten leafs enough so we have not been able to see fluorescence.

30Jun
  • June 30, 2016

Orange DNA Genome Extraction protocol href

  • href=" T"> Take from the glycerinates of GoldenBraid Collection:


Plasmid GB Code
href="Prom">Promoter 35s:Cas9:nopaline synthase terminator (Tnos) GB0639
Luciferase (Luc) in pUPD2 GB0096
Tnos in pUPD2 GB0037


01Jul
  • July 01, 2016

. Plasmid Mini Kit I, Q(capless) Spin of:

  • href=" "> Promoter 35s:Cas9 : Tnos


t succeed. Repeat Luc and Tnos cultures.


Primers IG16JUN01 and IG16JUN02 have arrived.

Finish orange DNA Genome Extraction and check DNA concentration with NanoDrop. Concentration is very low so extraction will be done again.

02Jul
  • July 02, 2016

. Plasmid Mini Kit I, Q(capless) Spin of:

  • href=" "> Luc in pUPD2

    • href=" "> Tnos in pUPD2


    • href=" C"> Check orange DNA genome concentration with NanoDrop.


    L)
    Clemenules1 3153.8
    Clemenules 2 4527.9


    • href=" P"> Perform a PCR to bind linker with luciferase:

    L) Program
    LuciferasepUPD 1 Temperature Time
    Buffer HF 10 98^C 5 minutes
    dNTPs 2 98^C 35x 30 seconds
    IG16JUN01 2.5 70^C 35x 30 seconds
    IG16JUN02 2.5 72^C 35x 1 minute 30 seconds
    Taq phusion 0.5 72^C 10 minutes


04Jul
  • July 04, 2016


Run electrophoresis gel of Clemenules DNA (agarose 1%). 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.

Rice DNA Genome Extraction Protocol href

Run electrophoresis gel of Luciferase PCR product. 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.

Ligate Reaction --> Linker:Luciferase into a pUPD2

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