IGEM:Yale/2010/Notebook/2010/07/08

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Ligation Efforts Continue

 * In addition to trying the triple-ligation strategy started on 7/7 (ligation attemp #3) will try trouble-shooting the ligation strategy used in attempts #1 and #2. In particular, will modify digestion protocol and test EcoRI and XbaI (older enzymes) for activity.

Ligation Attempt #3 (Triple Ligation)
Ran triple ligation all day at room temperature and stored at 4°C. Ligation contents were as follow to have a 1:1:1 stoichiometric ratio of all three components.

Ligation Attempt #4
Concerns about enzyme activity led to reversing the order of the serial digestion of B0015 background so that the first digestion is XbaI (low salt buffer) and the second is EcoRI (high salt buffer). Will also omit the pre-ligation PCR purification step.

Overnight digestion of ligation components
Set up the following digestions to run overnight at 37°C. The EcoRI was newly purchased as there was concern about the age of the previously used enzyme.

Wetlab work for this day is also recorded on pages 52 and 53 of the hard copy lab notebook.


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