Week9 8/8/10-8/14/10

From 2010.igem.org

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(New page: ---- ===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 --> should see 1...)
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===8/9/10===
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Bought Chitosan powder to use as a positive control for calcofluor white --> glowed bright blue/green when stained
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Stained (pMal-CHS3) ligation products for the presence of chitin --> ligation 1/tube 3 had slight fluorescence
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Used UV-vis Spectroscopy to measure chitin concentrations in ________
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Grew bakers yeast to test for presence of chitin
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Colony PCR on (pMal-CHS3)
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CP-LacPI part testing
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----
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===8/10/10===
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Ran gel for colony PCR no positives
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Ran PCR for CHS3 insert into pMal vector
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Stained varying concentrations of chitosan (.25g/ml, 25ug/ml, 5ug/ml, 1ug/ml) and (pMal-CHS3) cells --> cells showed no signs of chitin
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Digested __________ (sean)
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Ligated (CP-LacPi)-(GFP) in Tet backbone
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Made LB minimum media for electroporated cells
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Miniprepped circular backbone plasmid
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----
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===8/11/10===
===8/11/10===

Revision as of 18:25, 16 October 2010

Contents

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 in ________

Grew bakers yeast to test for presence of chitin

Colony PCR on (pMal-CHS3)

CP-LacPI part testing

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

Digested __________ (sean)

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 --> should see 1 2500kb band

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

Cycles 1 of File:MAGE for tqsA Knockouts.

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