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

From 2012.igem.org

(Difference between revisions)
Line 340: Line 340:
<li>0.5 ul BSA</li>
<li>0.5 ul BSA</li>
<li>0.5 ul EcoRI-HF</li>
<li>0.5 ul EcoRI-HF</li>
-
<li>0.5 ul PstI</li>
+
<li>0.5 ul SpeI</li>
-
<li>19 ul dH20 (0,5 ul less when using BSA</li>
+
<li>0.5 ul DpnI (Used to digest any template DNA from production)</li>
-
<li></li>
+
<li>19 ul dH20 (0,5 ul less when using BSA)</li>
</ul>
</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>  
</ol>  
</ol>
</ol>

Revision as of 23:28, 15 September 2012

iGEM TEAM ::: SDU-DENMARK courtesy of NIAID


mRNA Isolation PCR Miniprep Check Digest
3A-Assembly Content Content Content
Content Content Content Content

3A-Assembly -Ligation of 2 parts into a vector

  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

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