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Team:IvyTech-South Bend/Notebook
From 2012.igem.org
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<a href="#">Lab Notebook: See our weekly lab work</a>
June 18, 2012 Week 1. Staging and presentation of project storming to Ivy Tech advisory board. Parts research and descriptions.
June 25, 2012 Week 2. Continuation of parts research including promoters & killswitch screening and selction. Constitutive PTeT promoter, RBS, double terminator.
July 2, 2012 Week 3. Checked cultures and paired plates. Continuation of parts research and construction
July 9, 2012 Week 4. Oops Event!!! Protocol correction due to low yield or transformation. Killswitch transformation was redone (serial dilution test for transformation efficiency) Kamamiosin proves to be better selection for transformation for the psb1Ak3 plasmid backbone.
Pulled K190015 because its an arsenic sensitive promoter ẅ RFP
Transformed K190015 on Psb1A13
July 16, 2012 Week 5.
Propped Test promoter and transferred to Arsenic inducible promoter J33201 with Ars R repressor binding site. To be able to test for decreasing sensitivity to Arsenic.
July 23, 2012 Week 6.
Redid transformation on electrocompetent eColi cells. Results yielded pink & white colonies. Expanded the colonies and purfied K190015
-PINK Hypothesized to be succesful transformation with Rtt
-WHITE Unsure, proceeded to test in wk. 7
August 1, 2012 Week 7.
To test sensitivity of k190015; including pink and white colonies, ran 96 well and stimulating with dilute Arsenic sol. Results were inconclusive.
August 13, 2012 Week 9.
Ran gel to confirm band length of suspected parts, reran the 96 well plates yielding better results.
August 20, 2012 Week 10. To confirm S33201
August27, 2012 Week 11. Extract K190015 10ul, add DNA to ecom cells(5ul) into a tube, add to cuvette and flick to micropulsar. Take reading from BioRad and add soc immediately. 950ul of soc into cuvette, extract all contents and place into tubes. Repeat 3 times. Put in incubator for 1 hr@30°C, make 3 serial dilutions. Once serial dilution is complete spread on the amp plates, put streaked plates in incubator. The plates we poured were less than 10% red, the rest were white. Total of 64hrs. for growth to appear.
