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

From 2010.igem.org

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===Week9 Highlights===
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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.
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===8/9/10===
===8/9/10===
Bought Chitosan powder to use as a positive control for calcofluor white --> glowed bright blue/green when stained
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  
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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Used UV-vis Spectroscopy to measure chitin concentrations  
Grew bakers yeast to test for presence of chitin
Grew bakers yeast to test for presence of chitin
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Colony PCR on (pMal-CHS3)
Colony PCR on (pMal-CHS3)
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CP-LacPI part testing
 
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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
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)
 
Ligated (CP-LacPi)-(GFP) in Tet backbone
Ligated (CP-LacPi)-(GFP) in Tet backbone
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Miniprepped circular backbone plasmid
Miniprepped circular backbone plasmid
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===8/11/10===
===8/11/10===
Digested and ran a gel of CHS3 and backbone plasmids (C,T,K,A)
Digested and ran a gel of CHS3 and backbone plasmids (C,T,K,A)
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Tested restriction enzymes by digesting circular plasmids with each enzyme individually --> should see 1 2500kb band
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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.
Organized the DNA samples in our -20.
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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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Cycles 1 of [[Image:MAGE for tqsA Knockouts]].
 
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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