IGEM:Yale/2010/Notebook/2010/07/09

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Ligation Attempt #4
Followed with 20 minute heatkill at 80°C.
 * Removed both overnight digestions, the XbaI digestion of B0015 and the EcoRI and SpeI digestion of phsABC, from 7/8 from the incubator.
 * Heatkilled enzymes of phsABC digest with 20 minutes at 80°C.
 * Ran PCR purification protocol on XbaI digestion using microcentrifuge. In order to give a concentrated sample, eluted in only 35 μL of EB Buffer that had been heated at 50°C.  Resulting concentration could not be determined as there was residual ethanol contamination.
 * Ran EcoRI digestion of purified XbaI digest product at 37 °C, with components as follows. Digestion was run for an hour before addition of CIP, after which it was run for another hour.
 * In order to set up ligation, need concentration of B0015, but ethanol contaminant continues to preclude measurement. Will estimate that the purification between digestion steps resulted in a 50% loss of DNA, leaving behind a 10 ng/μL concentration. Will also, as usual, run different ligation trials with different ratios of insert to vector, which should help combat any issues if this estimate is inaccurate.
 * Set up the following ligation trials, with stoichiometric ratios of insert to vector based on B0015 concentration estimate. Ran the ligations for 20 minutes at room temperature.


 * Following ligation, transformed 1 μL of each ligation reaction into commercial grade cells according to standard transformation protocol. Also transformed triple ligation reaction.

Enzyme Activity Test
To test enzyme activity, will do single digests of B0015 with enzymes in question and then run them on a gel to see if the plasmid linearized. XbaI and EcoRI are old, so will test them first. For XbaI activity test, simply set aside 10 μL of digestion solution from overnight digestion. Testing of EcoRI required additional digests. Digests using both old and new EcoRI samples were run for two hours at 37 °C with the following components:

Followed with 20 minute heatkill at 80°C. Based on the results, all enzymes appeared to work, as they successfully created a linearized 3.2 kb fragment.
 * Loaded all three enzyme activity tests on a 1.0% agarose gel and ran them at 90 V versus a 1 kb ladder and circular B0015 plasmid.

Wetlab work for this day is also recorded on pages 54-57 of the hard copy lab notebook.


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