Difference between revisions of "Team:Ionis Paris/22 09 16"

 
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                                    <h2 class="blog_topHd"> <font color =”#279AD3”>Mini prep: on DH5⍺ transformed with BBA and BBB</font></h2>
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                                    Mini prep: on DH5⍺ transformed with BBA and BBB
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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 BBA and BBB plasmids extracted from bacterial mini-cultures in order to sequence them.</p>
 
             <p>Purification and quantification of BBA and BBB 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>28 Mini-cultures of bacteria transformed with BBA (n°1 to 14) and BBB (n°1 to 14) realized the 21/09 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube).<br/>
 
<p>28 Mini-cultures of bacteria transformed with BBA (n°1 to 14) and BBB (n°1 to 14) realized the 21/09 (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 on <a href="https://www.qiagen.com/fr/resources/resourcedetail?id=331740ca-077f-4ddd-9e5a-2083f98eebd5&lang=en"><font color = "DeepPink">thislink</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 on <a href="https://www.qiagen.com/fr/resources/resourcedetail?id=331740ca-077f-4ddd-9e5a-2083f98eebd5&lang=en"><font color = "DeepPink">thislink</font></a>).</p>
  
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<h3>Miniprep : </h3>
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<h5><font color =”#3CB5E1”>Miniprep : </font></h5>  
 
<ol>
 
<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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            <h3>Bacteria storage : </h3>
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          <h5><font color =”#3CB5E1”>Bacteria storage : </font></h5>  
 
<p>1.  Add 100 µL of glycerol 50% to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf.</p>
 
<p>1.  Add 100 µL of glycerol 50% to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf.</p>
 
         <li><p>42 tubes of BBA (3 per mini-cultures)</p></li>
 
         <li><p>42 tubes of BBA (3 per mini-cultures)</p></li>
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Latest revision as of 02:49, 20 October 2016

Mini prep: on DH5⍺ transformed with BBA and BBB

Objectives

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

Materials

28 Mini-cultures of bacteria transformed with BBA (n°1 to 14) and BBB (n°1 to 14) realized the 21/09 (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 on thislink).

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.

Bacteria storage :

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

  • 42 tubes of BBA (3 per mini-cultures)

  • 42 tubes of BBB (3 per mini-cultures)

    • 2. Store at -80°C.

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