Team:Calgary/Notebook/Protocols/GibsonAssembly

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<li>Assembly fragments were designed to have 35-40bp overhanging regions of
<li>Assembly fragments were designed to have 35-40bp overhanging regions of
complementary in the primers</li>
complementary in the primers</li>
-
<li>PCR fragments were generated using a <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/KapaPCR">KAPA</a> HiFi polymerase reaction. Fragments were
+
<li>PCR fragments were generated using a <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/KapaPCR" target="_blank">KAPA</a> HiFi polymerase reaction. Fragments were
-
verified on an <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/AgaroseGelElectrophoresis">agarose gel</a> and treated with DpnI restriction enzyme</li>
+
verified on an <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/AgaroseGelElectrophoresis" target="_blank">agarose gel</a> and treated with DpnI restriction enzyme</li>
<li>The following reaction was set up: 0.5 pmols of linear fragments, at
<li>The following reaction was set up: 0.5 pmols of linear fragments, at
-
least two fold excess of inserts and 75ng of vector backbone; 10uL of 2x
+
least two fold excess of inserts and 75ng of vector backbone; 10μL of 2x
-
NEB Gibson assembly master mix; H2O to bring entire reaction to 20uL</li>
+
NEB Gibson assembly master mix; water to bring entire reaction to 20μL</li>
-
<li>Gibson reaction was run in a thermocycler at 50C for 1 hour</li>
+
<li>Gibson reaction was run in a thermocycler at 50°C for 1 hour</li>
-
<li>2uL of the above reaction was transformed into NEB 5-alpha competent <i>E.coli</i></li>
+
<li>2μL of the above reaction was transformed into NEB 5-alpha competent <i>E.coli</i></li>
-
<li>Cells on plates were subsequently screen using standard <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/ColonyPCR">colony PCR</a> and
+
<li>Cells on plates were subsequently screened using standard <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/ColonyPCR" target="_blank">colony PCR</a> and
-
<a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/VerificationDigest"> verification digest </a> methods</li>
+
<a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/VerificationDigest" target="_blank"> verification digest </a> methods</li>
</ol>
</ol>

Latest revision as of 00:53, 28 September 2013

Gibson Assembly

Protocol

  1. Assembly fragments were designed to have 35-40bp overhanging regions of complementary in the primers
  2. PCR fragments were generated using a KAPA HiFi polymerase reaction. Fragments were verified on an agarose gel and treated with DpnI restriction enzyme
  3. The following reaction was set up: 0.5 pmols of linear fragments, at least two fold excess of inserts and 75ng of vector backbone; 10μL of 2x NEB Gibson assembly master mix; water to bring entire reaction to 20μL
  4. Gibson reaction was run in a thermocycler at 50°C for 1 hour
  5. 2μL of the above reaction was transformed into NEB 5-alpha competent E.coli
  6. Cells on plates were subsequently screened using standard colony PCR and verification digest methods

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