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

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      <a id="ligationreactioncohesive"></a>
 
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<div class="container">
 
           <div class="col-md-12">
 
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             <h2 class="featurette-heading">
 
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             <h1>Plasmid extraction </h1>
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             <h1>Ligation reaction (Cohesive ends)</h1>
 
             <br><br>
 
             <br><br>
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           <p>PureLink® Quick Plasmid Miniprep Kit-Life Technologies</p>
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           <p>ref: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202</p>
 
             <br>
 
             <br>
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             <h2>Methodology</h2>
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            <br><br>
 +
             <h2>Materials</h2> 
 +
            <br>
 +
          <ul>
 +
                <li>Vector DNA digested;</li>
 +
                <li>Insert DNA digested;</li>
 +
                <li>10X T4 DNA Ligase Buffer* (Thermo Scientific);</li>
 +
                <li>T4 DNA Ligase (Thermo Scientific);</li>
 +
                <li>Nuclease free water.</li>
 +
            <h3>Methodology</h3>
 
           <br>
 
           <br>
 
           <ul>
 
           <ul>
−
                 <li>Cell Growth</li>
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                 <li>Set up the following reaction in a microcentrifuge tube on ice:</li><br>
−
                      <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>
+
              <img src=https://static.igem.org/mediawiki/2015/d/db/BrasilUSPtabelaprotocols.png width=¨400¨/>
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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>
+
                       <ul>* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.</ul>
−
                <li>Lysis</li>
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                       <ul>q.s. = quantum sufficit</ul>
−
                       <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.
+
                       <ul>** ng of insert = (kb of insert) /(kb of vector x ng of vector (50 ng usually) x ratio. The ratio was considered to be 3, but it can vary according to the vector</ul>
−
</ul>
+
                 <li>For cohesive ends, incubate at 22°C for 3 hours + 16°C for 9 hours.</li>
−
                <li>Neutralization</li>
+
−
                       <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.
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</ul>
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             </ul>
 
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      <a id="agarosegeleletro"></a>
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          <div class="col-md-12">
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            <h2 class="featurette-heading">
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            <h1>Agarose Gel Electrophoresis </h1>
 +
            <br>
 +
            <h2>Materials</h2> 
 +
            <br>
 +
          <ul>
 +
                <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>
 +
                <li>Sterile liquid LB media (SIGMA-ALDRICH<sup>®</sup>);</li>
 +
                <li>Competent DH5α cells (Novagen) prepared through heat shock with calcium chloride;</li>
 +
                <li>Plasmidial DNA.</li>
 +
            </ul>
 +
            <br>
 +
            <h2>Methodology</h2>
 +
          <br>
 +
          <ul>
 +
                <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>
 +
                <li>Add 200 μL of liquid LB;</li>
 +
                <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>
 +
                <li>Incubate overnight (14- 16h) at 37°C.</li>
 +
            </ul>
 +
            <br>
 +
    </h2>
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 +
          </div>
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Revision as of 23:49, 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.

Digestion of plasmidial DNA


Materials


  • Plasmidial DNA;
  • Restriction Enzyme 1: EcoRI or XbaI (FastDigest Thermo Scientific);
  • Restriction Enzyme 2: SpeI or PstI (FastDigest Thermo Scientific);
  • FastDigest Buffer (Thermo Scientific);
  • Nuclease free water.

Methodology


  • On ice, prepare the following mixture in a microtube:
      • 500 -1000 ng of plasmidial DNA
      1 μL of Restriction Enzyme 1
      1 μL of Restriction Enzyme 2 (if necessary)
      2 μL of 10x FastDigest Buffer
      Nuclease free water to complete 20 μL
  • Spin the mixture.
  • Incubate at 37°C for at least 3 hours.
  • Perform agarose gel electrophoresis to confirm the results

Ligation reaction (Cohesive ends)



ref: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202




Materials


  • Vector DNA digested;
  • Insert DNA digested;
  • 10X T4 DNA Ligase Buffer* (Thermo Scientific);
  • T4 DNA Ligase (Thermo Scientific);
  • Nuclease free water.

    • Methodology


    • Set up the following reaction in a microcentrifuge tube on ice:

        • * The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
        q.s. = quantum sufficit
        ** ng of insert = (kb of insert) /(kb of vector x ng of vector (50 ng usually) x ratio. The ratio was considered to be 3, but it can vary according to the vector
    • For cohesive ends, incubate at 22°C for 3 hours + 16°C for 9 hours.

Agarose Gel Electrophoresis


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.

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