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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; 10μL of 2x | least two fold excess of inserts and 75ng of vector backbone; 10μL of 2x | ||
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<li>Gibson reaction was run in a thermocycler at 50°C for 1 hour</li> | <li>Gibson reaction was run in a thermocycler at 50°C for 1 hour</li> | ||
<li>2μL 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 | + | <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
Gibson Assembly
Protocol
- 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
