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__NOTOC__ ===Week9 Highlights=== We bought chitosan powder form a local health store to use as a positive control for chitin staining. We ran a colony PCR on pMAL-CHS3 and found no positive results. We began MAGE knockouts of tqsA. Discovered that our Holin1, Holin2, pMAL-CHS3, and CP-LacPi parts were faulty. We also decided to use a rhodamine-conjugated chitin probe as an alternative chitin stain because calcofluor white resulted in excess background. ---- ===8/9/10=== Bought Chitosan powder to use as a positive control for calcofluor white --> glowed bright blue/green when stained Stained (pMal-CHS3) ligation products for the presence of chitin --> ligation 1/tube 3 had slight fluorescence Used UV-vis Spectroscopy to measure chitin concentrations Grew bakers yeast to test for presence of chitin Colony PCR on (pMal-CHS3) ---- ===8/10/10=== Ran gel for colony PCR no positives Ran PCR for CHS3 insert into pMal vector Stained varying concentrations of chitosan (.25g/ml, 25ug/ml, 5ug/ml, 1ug/ml) and (pMal-CHS3) cells --> cells showed no signs of chitin Ligated (CP-LacPi)-(GFP) in Tet backbone Made LB minimum media for electroporated cells Miniprepped circular backbone plasmid ---- ===8/11/10=== Digested and ran a gel of CHS3 and backbone plasmids (C,T,K,A) Tested restriction enzymes by digesting circular plasmids with each enzyme individually --> saw 1 2500kb band which was expected Organized the DNA samples in our -20. ---- ===8/12/10=== Weekly meeting *Went over our gels --> identified faulty parts (holin1, holin2, CHS3-pMal, CP-LacPI) *Plan to re-kit to stock and reassemble Decided to use an alternative stain because calcofluor results in too much background and our confocal microscope can not excite in the UV spectrum ---- ===8/13/10=== Methanol fixation of yeast and E.Coli cells --> stained with rhodamine-conjugated chitin probe (allowed to incubate overnight) Competency of ChiA knockouts = 2.5x10^7 and 5.0x10^6 ----

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