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

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      <a id="digestion"></a>
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          <div class="col-md-12">
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            <h2 class="featurette-heading">
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            <h1>Digestion of plasmidial DNA</h1>
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            <br>
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            <h2>Materials</h2> 
 +
            <br>
 +
          <ul>
 +
                <li>Plasmidial DNA;</li>
 +
                <li>Restriction Enzyme 1: EcoRI or XbaI (FastDigest Thermo Scientific);</li>
 +
                <li>Restriction Enzyme 2: SpeI or PstI (FastDigest Thermo Scientific);</li>
 +
                <li>FastDigest Buffer (Thermo Scientific);</li>
 +
                <li>Nuclease free water.</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>
 +
                <li>On ice, prepare the following mixture in a microtube:</li>
 +
                      <ul>500 -1000 ng of plasmidial DNA</ul>
 +
                      <ul>1 μL of Restriction Enzyme 1</ul>
 +
                      <ul>1 μL of Restriction Enzyme 2 (if necessary)</ul>
 +
                      <ul>2 μL of 10x FastDigest Buffer</ul>
 +
                      <ul>Nuclease free water to complete 20 μL</ul>
 +
                <li>Spin the mixture.</li>
 +
                <li>Incubate at 37°C for at least 3 hours.</li>
 +
                <li>Perform agarose gel electrophoresis to confirm the results</li>
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            </ul>
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            <br>
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    </h2>
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          </div>
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            <h1>Plasmid extraction </h1>
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            <br><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>
 +
                <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.
 +
</ul>
 +
                <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.
 +
</ul>
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                <li>Washing</li>
 +
                      <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>
 +
            </ul>
 +
            <br>
 +
    </h2>
 +
 
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</div>
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      </div>
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{{:Team:Brasil-USP/Templates/Foot}}

Revision as of 23:08, 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

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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