User:Brian P. Josey/Notebook/2009/09/29
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Ligation of PBR 322 Unzipping Construct
Anthony taught me how to set up the ligation that he need today. To do this, we need 50μL of the total solution which includes six different components. First the water is added to the microcentrifuge tube, followed by 20μL of DNA, which is the one that we are studying and 1.026μL of an anchor, which is DNA designed to attach to the glass. To connect these two strands of DNA, we include a total of 4.00μL of an adapter, which is a strand of DNA that will attach the strand that we are studying to the strand attached to the glass. We add this in parts, starting with an original 1.00μL, and an additional 1.00μL every thirty minutes until reaching the total desired volume. The reason that we add this slowly is that if all of it was added at once, the strand we are studying and the anchors could both attach to the adapter, but not to eachother. We then add the buffer followed by the ligase. The buffer is added second to last because it contains ATP, and the ligase is added last because it is an enzyme that could be activated before we start the reaction.