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
Cycles 1 of [[Image:MAGE for tqsA Knockouts]].

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.


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)


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


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.


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


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