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Week9 8/8/10-8/14/10
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Competency of ChiA knockouts = 2.5x10^7 and 5.0x10^6 | Competency of ChiA knockouts = 2.5x10^7 and 5.0x10^6 | ||
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Latest revision as of 02:09, 27 October 2010
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
