Difference between revisions of "Team:Brasil-USP/Notebook/protocols"

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        <h1 style="border-bottom: 0px; margin: 0px 0px 25px 0px;">Protocols</h1>
 
        
 
        
 
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           <li><a href="#circuitassemblyprotocols">Circuit Assembly Protocols</a></li>
 
           <li><a href="#circuitassemblyprotocols">Circuit Assembly Protocols</a></li>
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           <li><a href="#sampleprepflowcyt">Flow Cytometer</a></li>   
 
           <li><a href="#sampleprepflowcyt">Flow Cytometer</a></li>   
 
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        <br />In order t
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            <h2 class="featurette-heading">
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            <h1>Calcium chloride transformation  with heat shock in Escherichia coli DH5α</h1>
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            <br>
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            <h2>Materials</h2>  
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            <br>
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          <ul>
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                <li>Sterile LB agar plate supplemented with the appropriate antibiotic (ampicillin 100 μg ml<sup>-1</sup> or chloramphenicol 34 μg ml<sup>-1</sup> - SIGMA-ALDRICH<sup>®</sup>);</li>
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                <li>Sterile liquid LB media (SIGMA-ALDRICH<sup>®</sup>);</li>
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                <li>Competent DH5α cells (Novagen) prepared through heat shock with calcium chloride;</li>
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                <li>Plasmidial DNA.</li>
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            </ul>
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            <br>
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            <h2>Methodology</h2>
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          <br>
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          <ul>
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                <li>Put the 0.5 mL microtube containing 50 μL competent cells aliquot on ice;</li>
 +
                <li>Add 20-50 ng of plasmidial DNA or 10  μL of ligation reaction to the competent cells. Mix by pipetting carefully;</li>
 +
                <li>Place the tube into a 42°C water bath for 2 min;</li>
 +
                <li>Return the tube to the ice for 5 min;</li>
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                <li>Add 200 μL of liquid LB;</li>
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                <li>Incubate at 37°C, 250 rpm for 45 min;</li>
 +
                <li> Plate the liquid LB containing the bacterial suspension on a LB agar plate with the appropriate antibiotic;</li>
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                <li>Incubate overnight (14- 16h) at 37°C.</li>
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            </ul>
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            <br>
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    </h2>
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            <h1>Plasmid extraction </h1>
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            <br>
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            <p>PureLink® Quick Plasmid Miniprep Kit-Life Technologies</p> 
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            <br>
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            <h2>Methodology</h2>
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          <br>
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          <ul>
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                <li>Cell Growth</li>
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                      <ul>After isolating a single colony from a LB agar plate, grow it in 6 mL of liquid LB within the appropriate antibiotic. Incubate overnight (14-16h) at 37°C in a shaking incubator.</ul>
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                <li>Resuspension</li>
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                      <ul>Pellet the overnight culture in a 2 mL microtube and discard the supernatant. Repeat this step until the total liquid culture is finished. Resuspend the cell pellet in 240 μL of resuspension buffer by vortexing.</ul> 
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                <li>Lysis</li>
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                      <ul>Add 250 μL of the Lysis buffer. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this period.
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</ul>
 +
                <li>Neutralization</li>
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                      <ul>Add 350 μL of the neutralization solution. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this time. Centrifuge at 16000 g for 10 minutes.
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</ul>
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                <li>Washing</li>
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                      <ul>ransfer the supernatant to a new 1.5 mL microtube with the resin. Be careful not to transfer the white pellet.
 +
Add 650 μL of Wash buffer. Centrifuge at 16000 g for 1 minute. Discard the supernatant. Centrifuge again for 2-4 min to remove ethanol remains.
 +
</ul>
 +
                <li>Elution of plasmidial DNA</li>
 +
                      <ul>Put the resin in a new 1.5 mL microtube. Add 50 μL of nuclease free water at 65°C. Centrifuge at 16000 g for 3 minutes and discard the resin. Store DNA at -20°C.
 +
</ul>
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            </ul>
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    </h2>
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{{:Team:Brasil-USP/Templates/Foot}}

Revision as of 15:57, 16 September 2015

Protocols

Notebook

Protocols

...

Calcium chloride transformation with heat shock in Escherichia coli DH5α


Materials


  • Sterile LB agar plate supplemented with the appropriate antibiotic (ampicillin 100 μg ml-1 or chloramphenicol 34 μg ml-1 - SIGMA-ALDRICH®);
  • Sterile liquid LB media (SIGMA-ALDRICH®);
  • Competent DH5α cells (Novagen) prepared through heat shock with calcium chloride;
  • Plasmidial DNA.

Methodology


  • Put the 0.5 mL microtube containing 50 μL competent cells aliquot on ice;
  • Add 20-50 ng of plasmidial DNA or 10 μL of ligation reaction to the competent cells. Mix by pipetting carefully;
  • Place the tube into a 42°C water bath for 2 min;
  • Return the tube to the ice for 5 min;
  • Add 200 μL of liquid LB;
  • Incubate at 37°C, 250 rpm for 45 min;
  • Plate the liquid LB containing the bacterial suspension on a LB agar plate with the appropriate antibiotic;
  • Incubate overnight (14- 16h) at 37°C.

Plasmid extraction


PureLink® Quick Plasmid Miniprep Kit-Life Technologies


Methodology


  • Cell Growth
      • After isolating a single colony from a LB agar plate, grow it in 6 mL of liquid LB within the appropriate antibiotic. Incubate overnight (14-16h) at 37°C in a shaking incubator.
  • Resuspension
      • Pellet the overnight culture in a 2 mL microtube and discard the supernatant. Repeat this step until the total liquid culture is finished. Resuspend the cell pellet in 240 μL of resuspension buffer by vortexing.
  • Lysis
      • Add 250 μL of the Lysis buffer. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this period.
  • Neutralization
      • Add 350 μL of the neutralization solution. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this time. Centrifuge at 16000 g for 10 minutes.
  • Washing
      • ransfer the supernatant to a new 1.5 mL microtube with the resin. Be careful not to transfer the white pellet. Add 650 μL of Wash buffer. Centrifuge at 16000 g for 1 minute. Discard the supernatant. Centrifuge again for 2-4 min to remove ethanol remains.
  • Elution of plasmidial DNA
      • Put the resin in a new 1.5 mL microtube. Add 50 μL of nuclease free water at 65°C. Centrifuge at 16000 g for 3 minutes and discard the resin. Store DNA at -20°C.

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