Difference between revisions of "Team:Ionis Paris/07 09 16"

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                                    <h4 class="blog_topHd">
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                                    <h2 class="blog_topHd"> <font color =”#279AD3”> Mini prep: on DH5⍺ transformed with BB12mut and BB123mut</font></h2>  
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                                      Mini prep: on DH5⍺ transformed with BB12mut and BB123mut
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                                    </h4>
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                                    <h4 class="blog_topHd">Objectives</h4>                      
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                                  <h3><font color =”94FAF1”> Objectives </font></h3>                    
 
             <p>Purification and quantification of BB12mut and BB123mut plasmids extracted from bacterial mini-cultures in order to sequence them.</p>
 
             <p>Purification and quantification of BB12mut and BB123mut plasmids extracted from bacterial mini-cultures in order to sequence them.</p>
  
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                                    <h4 class="blog_topHd">Materials</h4>
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                                  <h3><font color =”94FAF1”> Materials </font></h3>
 
<p>16 Mini-cultures of bacteria transformed with BB12 (3, 4, 5, 7, 8, 9, 11, 12) and BB123 (1, 2, 4, 5, 7, 10, 11, 13) realized the 06/09 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube).<br/>
 
<p>16 Mini-cultures of bacteria transformed with BB12 (3, 4, 5, 7, 8, 9, 11, 12) and BB123 (1, 2, 4, 5, 7, 10, 11, 13) realized the 06/09 (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>
 
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>
  
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                                     <h4 class="blog_topHd">Protocol</h4>
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                                     <h3><font color =”94FAF1”> Protocol </font></h3>  
  
  
 
<p>The 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"><font color = "DeepPink">here</font></a>).</p>   
 
<p>The 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"><font color = "DeepPink">here</font></a>).</p>   
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                                     <h5><font color =”#3CB5E1”>Miniprep:</font></h5>  
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                                          <h3>Miniprep:</h3>
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<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>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>
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<li><p>Add 250 μL Buffer P2 and mix by inverting the tube 6 times. The solution turns blue. </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>Add 350 μL Buffer N3 and mix by inverting the tube 6 times. The solution turns colorless.</p></li>
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<li><p></p>Centrifuge for 10 min at 13,000 rpm.</li>     
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<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>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 500 µL Buffer PB. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.</p></li>
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Latest revision as of 01:18, 20 October 2016

Mini prep: on DH5⍺ transformed with BB12mut and BB123mut

Objectives

Purification and quantification of BB12mut and BB123mut plasmids extracted from bacterial mini-cultures in order to sequence them.

Materials

16 Mini-cultures of bacteria transformed with BB12 (3, 4, 5, 7, 8, 9, 11, 12) and BB123 (1, 2, 4, 5, 7, 10, 11, 13) realized the 06/09 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube).
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.

Protocol

The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following the protocol given by the supplier (available here).

Miniprep:

  1. 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.

  2. Resuspend the pellet in 62.5 μL Buffer P1 and pool the 4 Eppendorf tubes into a unique tube.

  3. Add 250 μL Buffer P2 and mix by inverting the tube 6 times. The solution turns blue.

  4. Add 350 μL Buffer N3 and mix by inverting the tube 6 times. The solution turns colorless.

  5. Centrifuge for 10 min at 13,000 rpm.

  6. Load 800 μL supernatant from step 5 to the QIAprep 2.0 spin column. Centrifuge for 1 min and discard the flow-through.

  7. Add 500 µL Buffer PB. Centrifuge for 1 min at 13,000 rpm and discard the flow-through.

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

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

  10. Place the QIAprep 2.0 spin column in a clean 1.5 mL microcentrifuge tube.

  11. 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.

  12. Calculate the quantity of DNA with the Nanodrop.

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

Miniprep:

  1. Add 100 µL of glycerol 50% to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf.

    • 32 tubes of BB12mut (4 per mini-cultures)

    • 32 tubes of BB123mut (4 per mini-cultures)

  2. Store at -80°C.

    NB: No nanodrop quantification of samples was performed due to the departure of the machine.

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