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