Difference between revisions of "Team:Ionis Paris/Protocol 10"

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                                       <div class="blog_top">
 
                                       <div class="blog_top">
 
                               <h4 class="blog" style="margin-top:20px">
 
                               <h4 class="blog" style="margin-top:20px">
−
                                     Exponential Amplification</h4>
+
                                     Exponential amplification</h4>
 +
<p>Primers should be designed with 5´ ends annealing back-to-back. It is recommend to use the NEB online design software, NEBaseChanger™. </p>
 
                                     <p> Prepare the following reaction mix:</p>
 
                                     <p> Prepare the following reaction mix:</p>
  
−
                                <figure class="postImg waves-effect">
+
                             
−
                                    <img src="https://static.igem.org/mediawiki/2016/8/89/T--Ionis_Paris--Protocol10Table1.png" alt="">
+
<table>
−
                                </figure>
+
<tr>
 +
<td> Component  </td>
 +
<td>  Volume </td>
 +
<td> Final concentration </td>
 +
</tr>
 +
<tr>
 +
<td> Q5 Hot Start High-Fidelity 2X Master Mix  </td>
 +
<td> 12.5 µL  </td>
 +
<td> 1X  </td>
 +
</tr>
 +
<tr>
 +
<td> 10 μM Forward Primer  </td>
 +
<td> 1.25 μL  </td>
 +
<td>  0.5 μM </td>
 +
</tr>
 +
<tr>
 +
<td> 10 μM Reverse Primer  </td>
 +
<td>  1.25 μL </td>
 +
<td> 0.5 μM  </td>
 +
</tr>
 +
<tr>
 +
<td> Template DNA (1–25 ng/μl)  </td>
 +
<td> 1 μL  </td>
 +
<td> 1-25 ng  </td>
 +
</tr>
 +
<tr>
 +
<td> Nuclease-free water  </td>
 +
<td> 9 µL  </td>
 +
<td>  </td>
 +
</tr>
 +
</table>
 +
 
 
                                 <p> Perform a thermocycling as follows:</p>
 
                                 <p> Perform a thermocycling as follows:</p>
 
                                         <li>
 
                                         <li>
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                                             <p>Hold: 4°C</p>
 
                                             <p>Hold: 4°C</p>
 
                                         </li>
 
                                         </li>
−
                                        <li>
+
                                     
−
                                            <p>Qsp 30 µL RNAse-free H2O</p>
+
−
                                        </li>
+
  
 
                                 <h4 class="blog" style="margin-top:20px"> KLD Reaction</h4>
 
                                 <h4 class="blog" style="margin-top:20px"> KLD Reaction</h4>
 
                                 <p> Prepare the following reaction mix:</p>
 
                                 <p> Prepare the following reaction mix:</p>
−
<img src="https://static.igem.org/mediawiki/2016/2/27/T--Ionis_Paris--Protocol10Table2.png" alt="">
+
<table>
 +
<tr>
 +
<td> Component </td>
 +
<td> Volume  </td>
 +
<td>  Final concentration </td>
 +
</tr>
 +
<tr>
 +
<td>  PCR product  </td>
 +
<td>  1 µL </td>
 +
<td>  </td>
 +
</tr>
 +
<tr>
 +
<td>  2X KLD Reaction Buffer </td>
 +
<td> 5 µL  </td>
 +
<td>  1X </td>
 +
</tr>
 +
<tr>
 +
<td>  10X KLD Enzyme Mix </td>
 +
<td>  1 µL </td>
 +
<td>  1X </td>
 +
</tr>
 +
<tr>
 +
<td> Nuclease-free Water  </td>
 +
<td>  3 µL </td>
 +
<td>  </td>
 +
</tr>
 +
</table>
  
  
 
                                     <p> Incubate for 5 min at room temperature</p>
 
                                     <p> Incubate for 5 min at room temperature</p>
 +
 
                                 <h4 class="blog" style="margin-top:20px">Transformation</h4>
 
                                 <h4 class="blog" style="margin-top:20px">Transformation</h4>
−
                                 <p>Add 5 μl of KLD mix to 50 μl of chemically-competent cells.<br/>
+
                                 <p>Add 5μL of KLD mix to 50μL of chemically-competent cells.<br/>
 
Incubate on ice for 30 minutes.<br/>
 
Incubate on ice for 30 minutes.<br/>
 
Heat shock at 42°C for 30 seconds.<br/>
 
Heat shock at 42°C for 30 seconds.<br/>
 
Incubate on ice for 5 minutes.<br/>
 
Incubate on ice for 5 minutes.<br/>
−
Add 950 μl SOC, gently shake at 37°C for 1 hour.<br/>
+
Add 950μL SOC, gently shake at 37°C for 1 hour.<br/>
−
Spread 40–100 μl onto appropriate selection plate, incubate overnight at 37°C.<br/> </p>
+
Spread 40–100 μL onto appropriate selection plate, incubate overnight at 37°C.<br/> </p>
  
  
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                                     <li>
 
                                     <li>
 
                                         <a href="Protocol 12">
 
                                         <a href="Protocol 12">
−
                                             <span>Protocol 12 : Bacterial growth analysis</span>
+
                                             <span>Protocol 12: Cell Survival assay</span>
 
                                            
 
                                            
 
                                         </a>
 
                                         </a>

Revision as of 06:31, 17 October 2016

Protocol 10: Site directed Mutagenesis

Aim: Modify a given section of a plasmid

Exponential amplification

Primers should be designed with 5´ ends annealing back-to-back. It is recommend to use the NEB online design software, NEBaseChanger™.

Prepare the following reaction mix:

Component Volume Final concentration
Q5 Hot Start High-Fidelity 2X Master Mix 12.5 µL 1X
10 μM Forward Primer 1.25 μL 0.5 μM
10 μM Reverse Primer 1.25 μL 0.5 μM
Template DNA (1–25 ng/μl) 1 μL 1-25 ng
Nuclease-free water 9 µL

Perform a thermocycling as follows:

  • Initial denaturation: 98°C for 30s

  • 25 cycles of :
    - 98°C 10s
    - Provided Ta temperature 10-30s
    - 72°C; 20/30S per kB (amplification)

  • 72°C for 2 min

  • Hold: 4°C

    • KLD Reaction

    Prepare the following reaction mix:

    Component Volume Final concentration
    PCR product 1 µL
    2X KLD Reaction Buffer 5 µL 1X
    10X KLD Enzyme Mix 1 µL 1X
    Nuclease-free Water 3 µL

    Incubate for 5 min at room temperature

    Transformation

    Add 5μL of KLD mix to 50μL of chemically-competent cells.
    Incubate on ice for 30 minutes.
    Heat shock at 42°C for 30 seconds.
    Incubate on ice for 5 minutes.
    Add 950μL SOC, gently shake at 37°C for 1 hour.
    Spread 40–100 μL onto appropriate selection plate, incubate overnight at 37°C.