Revision as of 00:19, 28 September 2013

Gibson Assembly

Assembly fragments were designed to have 35-40bp overhanging regions of complementary in the primers
PCR fragments were generated using a KAPA HiFi polymerase reaction. Fragments were verified on an agarose gel and treated with DpnI restriction enzyme
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
Gibson reaction was run in a thermocycler at 50°C for 1 hour
2μL of the above reaction was transformed into NEB 5-alpha competent E.coli
Cells on plates were subsequently screened using standard colony PCR and verification digest methods

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 verified on an <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/AgaroseGelElectrophoresis">agarose gel</a> and treated with DpnI restriction enzyme</li>
 <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; water 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 50 degrees celsius 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 screened using standard <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/ColonyPCR">colony PCR</a> and
 <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/VerificationDigest"> verification digest </a> methods</li>