Difference between revisions of "Team:UCL/Experiments"
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<div id="ligation"><h2>Ligation protocol</h2> | <div id="ligation"><h2>Ligation protocol</h2> | ||
<ol> | <ol> | ||
+ | <li> Calculate the amount of insert DNA required to maintain 1:3 backbone:insert molar ratio. Use formula:<br/> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/9/9a/UCL_ligationformula.png.png">. For standard ligations use 50 ng of vector DNA, increase the amounts of DNA if unsuccessful.</li> | ||
+ | <li> Prepare the ligation mixture: | ||
+ | <ul> - required amount of insert DNA</ul> | ||
+ | <ul> - required amount of vector DNA </ul> | ||
+ | <ul> - 1 ul of T4 ligase </ul> | ||
+ | <ul> - 2 ul of 10 x ligase buffer </ul> | ||
+ | <ul> - add milliQ H2O up to 20 ul</ul> | ||
+ | </li> | ||
+ | <li>Incubate at 16C for 30 minutes</li> | ||
+ | <li>Heat inactivate by incubating at 80C for 20 minutes</li> | ||
+ | <li>Keep on ice until ready to proceed with transformation protocol</li> | ||
+ | |||
</ol> | </ol> | ||
</div> | </div> |
Revision as of 16:07, 5 August 2015