Team:Calgary/Notebook/Protocols/GibsonAssembly

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<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; 10uL 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 20uL</li>
<li>Gibson reaction was run in a thermocycler at 50C for 1 hour</li>
<li>Gibson reaction was run in a thermocycler at 50C for 1 hour</li>
<li>2uL of the above reaction was transformed into NEB 5-alpha competent <i>E.coli</i></li>
<li>2uL of the above reaction was transformed into NEB 5-alpha competent <i>E.coli</i></li>

Revision as of 23:10, 27 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; 10uL of 2x NEB Gibson assembly master mix; water to bring entire reaction to 20uL
  4. Gibson reaction was run in a thermocycler at 50C for 1 hour
  5. 2uL of the above reaction was transformed into NEB 5-alpha competent E.coli
  6. Cells on plates were subsequently screen using standard colony PCR and verification digest methods