Difference between revisions of "Team:Carnegie Mellon/Protocols"

 
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<body>
 
<body>
 
   
 
   
 +
    <div class="jumbotron">
 +
      <div class="container">
 +
        <div class = "title">Protocols.</div>
 +
        <p> How we did the things we did.</p>
 +
      </div>
 +
    </div>
 +
<center><a href="https://docs.google.com/document/d/1xSVhy_OLhlsD8rd0qir8nLXpgtiwlR-UD9LOXi_yQco/pub" style="font-size:20px;">Click here to view all of our protocols! </a></center>
 
<div id="flip">Click to slide the panel down or up</div>
 
<div id="flip">Click to slide the panel down or up</div>
 
<div id="panel">Hello world!</div>
 
<div id="panel">Hello world!</div>
 +
 +
 +
<div id = "flip_proteinextraction">His-Tag Soluble Protein Extraction</div>
 +
<div id = "panel_proteinextraction"><ol>
 +
  <li>Centrifuge 1.5 ml of culture for 1min full speed at 15000 rpm.</li>
 +
  <li>Remove supernatant.</li>
 +
  <li>Add 250 µl of extraction buffer (1% octyl-beta-thioglucoside in 10mM Tris-Cl, pH7.5).</li>
 +
  <li>Incubate at room temperature for 10 min.</li></ol>
 +
  <br>
 +
  <b>Wash Beads</b>
 +
  <ol><li>Remove 50 µl of Ni-NTA in 10 mM Trischloride bead solution and place in new tube for each purification.</li>
 +
  <li>Add 1 mL buffer (see table).</li>
 +
  <li>Centrifuge at 800 rpm for 30 seconds.</li>
 +
  <li>Remove supernatant.</li>
 +
  <li>Repeat 3 times and resuspend in 1 mL of Bead Wash Solution.</li>
 +
  <br>
 +
  <font size="4"> <ul>Seen below is the protocol we standardized and distributed to labs like the Citizen Science Center in an effort to gain more uniformity between data observed in different labs around the world. The back of the card has details on how to elute the protein off on the washed Ni-NTa beads. A download link for the JPEG can be found below the images.</ul> </font> <br>
 +
  <center> <font size="4"> <b>FRONT</b> </font> <br>
 +
  <image src="https://static.igem.org/mediawiki/2015/thumb/0/00/Protein_purification_1.jpg/800px-Protein_purification_1.jpg"> <br>
 +
    <font size="4"> <b>BACK</b> </font> <br>
 +
  <image src="https://static.igem.org/mediawiki/2015/thumb/d/d4/Protein_purification_2.jpg/800px-Protein_purification_2.jpg">
 +
  <br>
 +
  <a href = "https://static.igem.org/mediawiki/2015/0/0b/Protein_pur_card_FILES.zip"> PROTEIN PURIFICATION CARD JPEG </a>
 +
  </ol></div></center>
 +
 +
<!-- <div id = "flip_estrogen_sensor">Estrogen Sensor</div>
 +
<div id = "estrogen_sensor_p">Estrogen Sensor Protocol</div> -->
 +
 +
 +
 +
 +
<!-- ________________________________ Miniprep Protocol __________________________ -->
 +
<div id = "flip_miniprep">MiniPrep</div>
 +
<div id = "miniprep_p">
 +
<div><b>Minipreps of MACH Cells Expressing Flourescence</b></div>
 +
<div><b><u>Purpose:</u></b> To isolate plasmid DNA from MACH cells.</div>
 +
<p><b><u>Procedure:</u></b></p>
 +
<ol>
 +
<li>Set up overnight cultures for miniprep.</li>
 +
<li>Make the following reaction recipe:
 +
<ul><div class = "newbullet">
 +
<li>5 mL LB</li>
 +
<li> 5 µL Chlorophenical</li>
 +
<li>1 µL overnight colony</li>
 +
</div></ul></li>
 +
<li>Incubate in 37°C for 16-18 hours.</li>
 +
<li>Follow the <a target = "_blank" href = "https://tools.lifetechnologies.com/content/sfs/manuals/MAN0012655_GeneJET_Plasmid_Miniprep_UG.pdf">Life Technologies Miniprep Kit Protocol</a>.</li>
 +
</ol>
 +
</div>
 +
 +
<!-- __________________________ Restriction Enzyme Digest Protocol _____________ -->
 +
<div id = "flip_restriction_enzyme_digest">Restriction Enzyme Digestl</div>
 +
<div id = "restriction_enzyme_digest_p">
 +
 +
<div class = "title">Restriction Enzyme Digest</div>
 +
<div class = "procedure">Procedure:</div>
 +
<ol>
 +
<li>Prepare the following restriction enzyme digestion solution for J23108, J23109, J23111.</li>
 +
<table>
 +
<tr>
 +
<td><b><font color = "orange">Reagent</font></b></td>
 +
<td><b><font color = "orange">Amount (µL)</font></b></td>
 +
</tr>
 +
<tr>
 +
<td>10X Fast Digest Buffer</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td>Plasmid DNA</td>
 +
<td>12</td>
 +
</tr>
 +
<tr>
 +
<td>Restriction Enzyme XbaI</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Restriction Enzyme SpeI</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Water (to bring up to volume)</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td><b>Total Volume</b></td>
 +
<td><b>18</b></td>
 +
</tr>
 +
</table>
 +
<li>Digest at 37 °C for 1 hour.</li>
 +
<li>Follow the <a target = "_blank" href = "https://www.lifetechnologies.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/fastdigest-thermo-scientific.html">Life Technologies Restriction Enzyme Digest Protocol</a>.</li>
 +
</ol>
 +
</div><!-- restriction_enzyme_digest_p -->
 +
 +
 +
 +
<!-- __________________________ Agarose Gel Electrophoresis Protocol_____________ -->
 +
<div id = "flip_agarose_gel">Agarose Gel Electrophoresis</div>
 +
<div id = "agarose_gel_p">
 +
 +
<div class = "title">Aragose Gel Electrophoresis</div>
 +
<div class = "procedure">Procedure:</div>
 +
<ol>
 +
<li>1% aragose gel made of:
 +
<div class = "indentlist"><ul>50 mL 0.5X TAE</ul></div>
 +
<div class = "indentlist"><ul>0.5g agarose</ul></div>
 +
<div class = "indentlist"><ul>2.5 µL ethidium bromide</ul></div>
 +
</li>
 +
<li>Run at 114V.</li>
 +
</ol>
 +
</div><!-- agarose_gel_p -->
 +
 +
 +
 +
<!-- __________________________ Gel Extraction Protocol _____________ -->
 +
<div id = "flip_gel_extraction">Gel Extraction</div>
 +
<div id = "gel_extraction_p">
 +
 +
<div class = "title">Gel Extraction</div>
 +
<div class = "procedure">Procedure:</div>
 +
<ol>
 +
<li>Make 250-750 µl binding buffer (depending on mass of gel cut-out).</li>
 +
<li>Incubated at 42℃ until gel is melted.</li>
 +
<li>Follow Thermo Scientific's <a target = "_blank" href = "https://tools.lifetechnologies.com/content/sfs/manuals/MAN0012661_GeneJET_Gel_Extraction_UG.pdf">GeneJET Gel Extracton Protocol</a></li>
 +
</ol>
 +
</div><!-- gel_extraction_p -->
 +
 +
 +
<!-- __________________________ Ligation Protocol _____________ -->
 +
<div id = "flip_ligation">Ligation</div>
 +
<div id = "ligation_p">
 +
<div class = "title">Ligation</div>
 +
<div class = "procedure">Procedure:</div>
 +
<ol>
 +
<li>Make the following ligation reaction.</li>
 +
<table>
 +
<tr>
 +
<td><b><font color = "orange">Reagent</font></b></td>
 +
<td><b><font color = "orange">Amount (µL)</font></b></td>
 +
</tr>
 +
<tr>
 +
<td>Promoter DNA</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Insert DNA</td>
 +
<td>7</td>
 +
</tr>
 +
<tr>
 +
<td>Ligation Buffer</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Ligation Enzyme</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td><b>Total Volume</b></td>
 +
<td><b>10</b></td>
 +
</tr>
 +
</table>
 +
<li>Ligate for 10 minutes at room temperature.</li>
 +
<li>Put on ice until ready for transformation.</li>
 +
</ol>
 +
</div><!-- ligation_p -->
 +
 +
<!-- __________________________ Transformation Protocol _____________ -->
 +
<div id = "flip_transformation">Transformation</div>
 +
<div id = "transformation_p">
 +
<div class = "title">Transformation</div>
 +
<div class = "procedure">Procedure:</div>
 +
<ol>
 +
<li>Follow <a target = "_blank" href = "http://parts.igem.org/Help:Protocols/Transformation">iGEM's Transformation Protocol</a></li>
 +
</ol>
 +
</div><!-- transformation_p -->
  
 
</body>
 
</body>
  
 
</html>
 
</html>

Latest revision as of 19:05, 17 November 2015

Protocols.

How we did the things we did.

Click here to view all of our protocols!
Click to slide the panel down or up
Hello world!
His-Tag Soluble Protein Extraction
  1. Centrifuge 1.5 ml of culture for 1min full speed at 15000 rpm.
  2. Remove supernatant.
  3. Add 250 µl of extraction buffer (1% octyl-beta-thioglucoside in 10mM Tris-Cl, pH7.5).
  4. Incubate at room temperature for 10 min.

Wash Beads
  1. Remove 50 µl of Ni-NTA in 10 mM Trischloride bead solution and place in new tube for each purification.
  2. Add 1 mL buffer (see table).
  3. Centrifuge at 800 rpm for 30 seconds.
  4. Remove supernatant.
  5. Repeat 3 times and resuspend in 1 mL of Bead Wash Solution.

      6. Seen below is the protocol we standardized and distributed to labs like the Citizen Science Center in an effort to gain more uniformity between data observed in different labs around the world. The back of the card has details on how to elute the protein off on the washed Ni-NTa beads. A download link for the JPEG can be found below the images.
    FRONT

    BACK

    PROTEIN PURIFICATION CARD JPEG
MiniPrep
Minipreps of MACH Cells Expressing Flourescence
Purpose: To isolate plasmid DNA from MACH cells.

Procedure:

  1. Set up overnight cultures for miniprep.
  2. Make the following reaction recipe:
    • 5 mL LB
    • 5 µL Chlorophenical
    • 1 µL overnight colony
  3. Incubate in 37°C for 16-18 hours.
  4. Follow the Life Technologies Miniprep Kit Protocol.
Restriction Enzyme Digestl
Restriction Enzyme Digest
Procedure:
  1. Prepare the following restriction enzyme digestion solution for J23108, J23109, J23111.
    2. Reagent Amount (µL)
    10X Fast Digest Buffer 2
    Plasmid DNA 12
    Restriction Enzyme XbaI 1
    Restriction Enzyme SpeI 1
    Water (to bring up to volume) 2
    Total Volume 18
  2. Digest at 37 °C for 1 hour.
  3. Follow the Life Technologies Restriction Enzyme Digest Protocol.
Agarose Gel Electrophoresis
Aragose Gel Electrophoresis
Procedure:
  1. 1% aragose gel made of:
      50 mL 0.5X TAE
      0.5g agarose
      2.5 µL ethidium bromide
  2. Run at 114V.
Gel Extraction
Gel Extraction
Procedure:
  1. Make 250-750 µl binding buffer (depending on mass of gel cut-out).
  2. Incubated at 42℃ until gel is melted.
  3. Follow Thermo Scientific's GeneJET Gel Extracton Protocol
Ligation
Ligation
Procedure:
  1. Make the following ligation reaction.
    2. Reagent Amount (µL)
    Promoter DNA 1
    Insert DNA 7
    Ligation Buffer 1
    Ligation Enzyme 1
    Total Volume 10
  2. Ligate for 10 minutes at room temperature.
  3. Put on ice until ready for transformation.
Transformation
Transformation
Procedure:
  1. Follow iGEM's Transformation Protocol