Team:SDU-Denmark/labwork/Protocols/3A

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<table>
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<tr>
<tr>
<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/3A"><b>3A-Assembly</b></a></td>
<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/3A"><b>3A-Assembly</b></a></td>
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<td>Content</td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/colpcr">Colony-PCR </a></td>
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<td>Content</td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/Trans">Transformation</a></td>
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<td>Content</td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/gel">Gel-electrophoresis</a></td>
</tr>
</tr>
<tr>
<tr>
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<td>Content</td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/revtrans">Reverse Transcriptase</a></td>
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<td>Content</td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/mutagen">Mutagenesis</a></td>
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<td>Content</td>
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<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/pcrgelclean">PCR-,gel clean-up</a></td>
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<td>Content</td>
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</tr>
</tr>
</table>
</table>
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<h1> 3A assembly </h1>
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<h1> 3A-Assembly  </h1>
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<h2> Ligation of 2 parts into a vector</h2>
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 +
 
<p>
<p>
<ol>
<ol>
-
<li>PCR step; see PCR protocol</li>
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<li>PCR step; see PCR protocol</li>
-
<li>Digestion</li>
+
<li>Digestion</li>
-
<i>BSA is only needed for conventional restriction enzymes, NOT fast digest</i>
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<i>BSA is only needed for conventional restriction enzymes, NOT fast digest</i>
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<ol type="a">
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  <ol type="i">
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<li>Digest step for Plasmid Backbone</li>
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  <li>Digest step for Plasmid Backbone</li>
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</ol>
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  </br>
-
<li></li>
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<b>if sample comes from miniprep</b></br>
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<li></li>
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Enzyme Master Mix for Plasmid Backbone (25ul total)
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<li></li>
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  <ul type="square">
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<li></li>
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-
<li></li>
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    <li>5 ul Fastdigest greenbuffer / NEB Buffer 2</li>
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<li></li>
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    <li>0.5 ul BSA</li>
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(The procedure we used for mRNA isolation, is a modified version of the Qiagen RNeasy protocol for plant mRNA isolation.)
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    <li>0.5 ul EcoRI-H</li>
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<p>
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    <li>0.5 ul PstI</li>
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Small tubers from Helianthus tuberosus were washed with untreated water and cut in rough slices with an everyday boxcutter.</br>
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    <li>19 ul dH20 (0,5 ul less when using BSA)</li>
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The slices were distributed in cryotubes and weighed, the weights were noted down, and submerged in liquid nitrogen (flash-freeze at -196 °C). Flash-freezing is necessary to halt all RNase activity. </br>
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  </ul>
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RLT buffer is mixed at this point, for later use. For each mL RLT, add μL beta-mercaptoethanol. Do this in a fume cabinet, as mercaptoethanol smells really bad and is toxic!</br>
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</br>
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After 20 minutes 600mg is extracted from the cryotubes, the rest is stored for later use. The 600mg plant material is grinded to a fine dust using a mortar. It is important to keep the plant material frozen during the grinding, to keep the cell wall rigid enough to destroy it.</br>
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<b>If sample comes from PCR</b>
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The plant powder is transferred to a tube and dissolved in RLT buffer(450μL buffer op 100mg plant material). We used approx. 3mL for 600mg material.</br>
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</br>
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The solution is mixed using a vortex mixer.</br>
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-
500 μL of the solution is transfered to a 2mL treated with ultrasound to homogenize the content and further disrupt the cell wall. 500 μL is transfered to a 2mL tube as a control.</br>
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Enzyme Master Mix for Plasmid Backbone (25ul total)
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The samples are centrifuged at 14.500 rpm for 60 seconds to pellet out large organelles.
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<ul type="square">
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0,5 vol. 96-100% ethanol is added(250μL) to the sample, mixed by pipette. </br>
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-
Transfer the samples to spin-column (Qiagen RNeasy mini kit, for RNA isolation from animal cells) in a 2mL sampletube and do the following:
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<li>5 ul Fastdigest greenbuffer / NEB Buffer </li>
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<p>
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<li>0.5 ul BSA</li>
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-spin at 10.000 rmp for 15s, discard flowthrough
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<li>0.5 ul EcoRI-HF</li>
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add 700μL RW1 til spin column <p>
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<li>0.5 ul PstI</li>
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-spin at 10.000 rmp for 15s, discard flowthrough
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<li>0.5 ul DpnI (Used to digest any template DNA from productio</li>
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add 500μL RPE buffer til spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol)<p>
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<li>19 ul dH20 (0,5 ul less when using BSA</li>
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-spin at 10.000 rmp for 15s, discard flowthrough
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</ul>
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add another 500μL RPE buffer to the spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol)<p>
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</br>
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-spin at 10.000 rmp for 15s, discard flowthrough
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<b>Digest Plasmid Backbone</b>
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Transfer the columns to new 2mL centrifuge tubes. Centrifuge at full speed for 1 min.
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-
Transfer spin columns to new 1.5mL sample tubes <p>
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<ul>
-
-add 30-50μL RNase-free water <p>
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<li>Add 4 ul linearized plasmid backbone (25ng/ul for 100ng tota</li>
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-spin at 10.000 rpm for 60 seconds <p>
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<li>Add 4 ul of Enzyme Master Mix</li>
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Repeat the prior step once.
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<li>Digest 37C/30 min, heat kill Restriction Enzyme 80C/20 min</li>
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<p>
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</ul>
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The mRNA is now isolated in the 60-100μL at the bottom of the collection tubes
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</br>
-
<p>
+
<li>Digest step for Insert X:</li>
 +
</br>
 +
 
 +
<b>If sample comes from miniprep</b>
 +
</br>
 +
Enzyme Master Mix for Plasmid Backbone (25ul total)
 +
<ul>
 +
<li>5 ul Fastdigest greenbuffer / NEB Buffer</li>
 +
<li>0.5 ul BSA</li>
 +
<li>0.5 ul EcoRI-HF</li>
 +
<li>0.5 ul PstI</li>
 +
<li>19 ul dH20 (0,5 ul less when using BSA</li>
 +
 
 +
</ul>
 +
 
 +
<b>If sample comes from PCR</b>
 +
</br>
 +
Enzyme Master Mix for Plasmid Backbone (25ul total)
 +
<ul>
 +
<li>5 ul Fastdigest greenbuffer / NEB Buffer</li>
 +
<li>0.5 ul BSA</li>
 +
<li>0.5 ul EcoRI-HF</li>
 +
<li>0.5 ul SpeI</li>
 +
<li>0.5 ul DpnI (Used to digest any template DNA from production)</li>
 +
<li>19 ul dH20 (0,5 ul less when using BSA)</li>
 +
</ul>
 +
</br>
 +
<b>Digest Plasmid Backbone</b>
 +
    </br>
 +
<ul>
 +
<li>Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)</li>
 +
<li>Add 4 ul of Enzyme Master Mix</li>
 +
<li>Digest 37C/30 min, heat kill Restriction Enzyme 80C/20 min</li>
 +
 
 +
</ul>
 +
 
 +
<li>Digest step for Insert Y:</li>
 +
 
 +
 
 +
<b>If sample comes from miniprep</b>
 +
</br>
 +
Enzyme Master Mix for Plasmid Backbone (25ul total)
 +
<ul>
 +
<li>5 ul Fastdigest greenbuffer / NEB Buffer 2</li>
 +
<li>0.5 ul BSA </li>
 +
<li>0.5 ul EcoRI-HF </li>
 +
<li>0.5 ul PstI </li>
 +
<li>19 ul dH20 (0,5 ul less when using BSA) </li>
 +
</ul>
 +
 
 +
 
 +
<b>If sample comes from PCR</b>
 +
</br>
 +
Enzyme Master Mix for Plasmid Backbone (25ul total)
 +
<ul>
 +
<li>5 ul Fastdigest greenbuffer / NEB Buffer 2</li>
 +
<li>0.5 ul BSA</li>
 +
<li>0.5 ul Xbal</li>  
 +
<li>0.5 ul PstI</li>
 +
<li>0.5 ul DpnI (Used to digest any template DNA from production)</li>
 +
<li>19 ul dH20 (0,5 ul less when using BSA)</li>  
 +
 
 +
</ul>
 +
</br>
 +
<b>Digest Plasmid Backbone</b>
 +
    </br>
 +
<ul>
 +
<li>Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)</li>
 +
<li>Add 4 ul of Enzyme Master Mix</li>
 +
<li>Digest 37C/30 min, heat kill Restriction Enzyme 80C/20 min</li>
 +
</ul>
 +
  </ol>
 +
<li>Ligation step</li>
 +
 
 +
<ul>
 +
<li>Add 2ul of digested plasmid backbone (25 ng)</li>
 +
<li>Add 5:1 (Molar, NOT VOLUME)amount of EcoRI-HF SpeI digested fragment (Insert X)(< 3 ul)</li>
 +
<li>Add 5:1 (Molar, NOT VOLUME) amount of XbaI PstI digested fragment (Insert Y)(< 3 ul)</li>
 +
<li>Add 2 ul T4 DNA ligase buffer</li>
 +
<li>Add 0.5 ul T4 DNA ligase</li>
 +
<li>Add water to 10 ul</li>
 +
<li>Ligate 16C/30 min, heat kill 80C/20 min</li>
 +
   
 +
3A Assembly. The part B0034 and C0010 is referred to as X and Y
 +
</ol>
 +
 
 +
</br>
 +
</br>
 +
<img src="http://partsregistry.org/wiki/images/4/42/3AAssembly.png" alt="" width="680px" />
 +
</p>

Latest revision as of 21:09, 26 September 2012

iGEM TEAM ::: SDU-DENMARK courtesy of NIAID


mRNA Isolation PCR Miniprep Check Digest
3A-Assembly Colony-PCR Transformation Gel-electrophoresis
Reverse Transcriptase Mutagenesis PCR-,gel clean-up

3A-Assembly

  1. PCR step; see PCR protocol
  2. Digestion
    3. BSA is only needed for conventional restriction enzymes, NOT fast digest
    1. Digest step for Plasmid Backbone

      2. if sample comes from miniprep
      Enzyme Master Mix for Plasmid Backbone (25ul total)
      • 5 ul Fastdigest greenbuffer / NEB Buffer 2
      • 0.5 ul BSA
      • 0.5 ul EcoRI-H
      • 0.5 ul PstI
      • 19 ul dH20 (0,5 ul less when using BSA)

      If sample comes from PCR
      Enzyme Master Mix for Plasmid Backbone (25ul total)
      • 5 ul Fastdigest greenbuffer / NEB Buffer
      • 0.5 ul BSA
      • 0.5 ul EcoRI-HF
      • 0.5 ul PstI
      • 0.5 ul DpnI (Used to digest any template DNA from productio
      • 19 ul dH20 (0,5 ul less when using BSA

      Digest Plasmid Backbone
      • Add 4 ul linearized plasmid backbone (25ng/ul for 100ng tota
      • Add 4 ul of Enzyme Master Mix
      • Digest 37C/30 min, heat kill Restriction Enzyme 80C/20 min

    2. Digest step for Insert X:

      3. If sample comes from miniprep
      Enzyme Master Mix for Plasmid Backbone (25ul total)
      • 5 ul Fastdigest greenbuffer / NEB Buffer
      • 0.5 ul BSA
      • 0.5 ul EcoRI-HF
      • 0.5 ul PstI
      • 19 ul dH20 (0,5 ul less when using BSA
      If sample comes from PCR
      Enzyme Master Mix for Plasmid Backbone (25ul total)
      • 5 ul Fastdigest greenbuffer / NEB Buffer
      • 0.5 ul BSA
      • 0.5 ul EcoRI-HF
      • 0.5 ul SpeI
      • 0.5 ul DpnI (Used to digest any template DNA from production)
      • 19 ul dH20 (0,5 ul less when using BSA)

      Digest Plasmid Backbone
      • Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
      • Add 4 ul of Enzyme Master Mix
      • Digest 37C/30 min, heat kill Restriction Enzyme 80C/20 min
    3. Digest step for Insert Y:
      4. If sample comes from miniprep
      Enzyme Master Mix for Plasmid Backbone (25ul total)
      • 5 ul Fastdigest greenbuffer / NEB Buffer 2
      • 0.5 ul BSA
      • 0.5 ul EcoRI-HF
      • 0.5 ul PstI
      • 19 ul dH20 (0,5 ul less when using BSA)
      If sample comes from PCR
      Enzyme Master Mix for Plasmid Backbone (25ul total)
      • 5 ul Fastdigest greenbuffer / NEB Buffer 2
      • 0.5 ul BSA
      • 0.5 ul Xbal
      • 0.5 ul PstI
      • 0.5 ul DpnI (Used to digest any template DNA from production)
      • 19 ul dH20 (0,5 ul less when using BSA)

      Digest Plasmid Backbone
      • Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
      • Add 4 ul of Enzyme Master Mix
      • Digest 37C/30 min, heat kill Restriction Enzyme 80C/20 min
  3. Ligation step
    • Add 2ul of digested plasmid backbone (25 ng)
    • Add 5:1 (Molar, NOT VOLUME)amount of EcoRI-HF SpeI digested fragment (Insert X)(< 3 ul)
    • Add 5:1 (Molar, NOT VOLUME) amount of XbaI PstI digested fragment (Insert Y)(< 3 ul)
    • Add 2 ul T4 DNA ligase buffer
    • Add 0.5 ul T4 DNA ligase
    • Add water to 10 ul
    • Ligate 16C/30 min, heat kill 80C/20 min
      • 3A Assembly. The part B0034 and C0010 is referred to as X and Y


Retrieved from "http://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/3A"