Team:TU-Delft/Protocol 8

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<div style="margin-left:30px;margin-right:30px; width:900px;float:left;"> 
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<h2 align="center">Protocols</h2>
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<a name="protocol_8"></a>
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Our project deals with <i>E.coli</i> cells which sense Auto-inducing peptides (AIPs) from the <i>Staphylococcus aureus</i> and starts producing Antimicrobial peptides in order to kill the <i>Staphylococcus aureus</i>. Different protocols used during the project are described below.
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  <li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_1" style="text-decoration: none""><font color="#0080FF" size="3">Transforming Parts from Distribution kit</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol2" style="text-decoration: none""><font color="#0080FF" size="3">Growing the Single Colonies from the Agar Plates</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_3" style="text-decoration: none""><font
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color="#0080FF" size="3"> Making glycerol stocks</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_4" style="text-decoration: none""><font
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color="#0080FF" size="3"> Miniprep Protocol</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_5" style="text-decoration: none""><font
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color="#0080FF" size="3"> Restriction digestion</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_6" style="text-decoration: none""><font
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color="#0080FF" size="3"> Ligation</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_7" style="text-decoration: none"" ><font
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color="#0080FF" size="3"> Gel Extraction Procedure</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_8" style="text-decoration: none""><font
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color="#0080FF" size="3">  PCR Purification</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_9" style="text-decoration: none""><font
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color="#0080FF" size="3">  Tricine Gels</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_10" style="text-decoration: none""><font color="#0080FF" size="3">General Peptide Production</font></a> </li>
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<h2 align="center">PCR Purification:</h2>
<h2 align="center">PCR Purification:</h2>
<h4 align="left">Requirements:</h4>
<h4 align="left">Requirements:</h4>
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1. Buffer PB
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1. Buffer PB <br>
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2. Pipettes
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2. Pipettes <br>
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3. Buffer PE  
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3. Buffer PE <br>
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4. Microcentrifuge tubes
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4. Microcentrifuge tubes <br>
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5. QIA quick spin columns
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5. QIA quick spin columns <br>
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<h4 align="left">Prodecure:</h4>
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<h4 align="left">Procedure:</h4>
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1. Add 5 volumes of Buffer PB to 1 volume of the sample. <br>
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2. Transfer the mixture to the QIA quick spin columns and centrifuge the column for 1 min at 13000 rpm. <br>
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<li>Add 5 volumes of Buffer PB to 1 volume of the sample.</li>
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3. Discard the flow through and place the spin column in the same collection tube.<br>
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<li>Transfer the mixture to the QIA quick spin columns and centrifuge the column for 1 min at 13000 rpm. </li>
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4. To wash, add 0.75 mL of Buffer PE to the column, and centrifuge for 1 min. Discard the flow through and place the column back in the same collection tube. <br>
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<li> Discard the flow through and place the spin column in the same collection tube.</li>
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5. Centrifuge the empty column for an additional 1 min to remove the remaining Buffer PE from the column. <br>
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<li>To wash, add 0.75 mL of Buffer PE to the column, and centrifuge for 1 min. Discard the flow through and place the column back in the same collection tube.</li>
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6. Now place the column in a fresh microcentrifuge tube. Add 50 µL of sterile milliQ water. <br>
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<li>Centrifuge the empty column for an additional 1 min to remove the remaining Buffer PE from the column. </li>
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7. Place the column in oven for 2 mins, then centrifuge again for 1 min at 13000 rpm. <br>  
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<li> Now place the column in a fresh microcentrifuge tube. Add 50 µL of sterile milliQ water. </li>
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8. Measure the sample on Nanodrop to get the concentration. <br>  
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<li>Place the column in oven for 2 mins, then centrifuge again for 1 min at 13000 rpm. </li>
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<li> Measure the sample on Nanodrop to get the concentration.</li>
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<a name="references"></a>
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<h2 align="center">References</h2>
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PCR Purification Kit Protocol<i>QIAquick Spin Handbook, July 2002 </i>.[Online]. Available From:
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<a href="http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAquickSpin_EN.pdf" style="text-decoration: none"" target="_blank">
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http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAquickSpin_EN.pdf</a>
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</li>
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</ol>
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</p>
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</html>
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Latest revision as of 15:34, 3 October 2013



Protocols

Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.



PCR Purification:

Requirements:

1. Buffer PB
2. Pipettes
3. Buffer PE
4. Microcentrifuge tubes
5. QIA quick spin columns

Procedure:

  1. Add 5 volumes of Buffer PB to 1 volume of the sample.
  2. Transfer the mixture to the QIA quick spin columns and centrifuge the column for 1 min at 13000 rpm.
  3. Discard the flow through and place the spin column in the same collection tube.
  4. To wash, add 0.75 mL of Buffer PE to the column, and centrifuge for 1 min. Discard the flow through and place the column back in the same collection tube.
  5. Centrifuge the empty column for an additional 1 min to remove the remaining Buffer PE from the column.
  6. Now place the column in a fresh microcentrifuge tube. Add 50 µL of sterile milliQ water.
  7. Place the column in oven for 2 mins, then centrifuge again for 1 min at 13000 rpm.
  8. Measure the sample on Nanodrop to get the concentration.

    9. References

    1. PCR Purification Kit ProtocolQIAquick Spin Handbook, July 2002 .[Online]. Available From: http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAquickSpin_EN.pdf

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