Team:Cornell/testing/notebook/wetlab/4

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Weekly Update
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Wet Lab - September

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    Week 1

    Conjugating nah into Shewy: The mysterious failure of past week's ligation lead to a redigestion (in case we run out of DNA) and religation of last week's digestions. Nah to oriT was ligated in ratios of 3:1 and 6:1 - when run on a gel, bands once more failed to appear... But wait! Epiphany! The team realized that we forgot to dephosphorylate the oriT backbone! After that silly mistake, 1:1, 3:1, and 6:1 ratios of nah:oriT were set up in a ligation mixture and electroporated into WM3064. Once again, slow growth and tiny colonies were observed - perhaps due to the strain of putting such a massive operon in a cell. 13 colonies were deemed large enough to try colony PCR, and 3 of them definitely showed bands that corresponded to the length of our construct. We set up liquid cultures for Miniprepping and once again observed slow growth. We tried subculturing - but still, general opaqueness. Miniprepping proceeded anyway, as we are a fairly optimistic bunch (and the cultures were by then over 24 hours old).

    Site-directed mutagenesis: After several more unsuccessful attempts, lengthy discussion among teammates, and consultation with several graduate advisors, the team decided to put this subproject on hold for the time being. Swati finished up with one last attempt, then joined Claire for fluorescence testing. That's all, folks! Daily Details

    Daily Details:

    September 1

    Conjugating nah into Shewy: Due to the mysterious failings of our attempts to ligate the nah operon, Tina tried to start fresh by re-digesting the nah operon-containing plasmid and the oriT-containing backbone.

    Synthetic River Media: Mark and Chie created synthetic river media based on information on the mineral content of the Athabasca River, and started overnight cultures of numerous strains to test how sodium lactate supplemented river water would grow the cells.

    Site-directed mutagenesis: Swati transformed both her mutagenic PCR and her DpnI digest into DH5a after desalting on a membrane. She also ran both of the aforementioned samples on a gel without purifying, in order to check if either the digestion or purification steps were problematic. The gel was yet again blank, suggesting that the PCR itself was the problem.

    September 2nd

    Conjugating nah into Shewy: Tina tried ligating an older digestion of the nah operon into the oriT backbone using a molar ratio or 3:1 and 6:1 for nah:backbone. She then de-salted and transformed the ligations into conjugation strain WM3064. Tina then ran a gel of the digested nah operon-containing plasmid and the oriT-containing backbone and extracted the nah operon and oriT backbone bands.

    Synthetic River Media: Mark set up a 96-well plate to test growth of the engineered strains in sodium lactate at varying concentrations, in synthetic river media.

    Site-directed mutagenesis:The third transformation attempt yielded no colonies on the plate of DpnI-digested DNA, but three colonies on the PCR only plate, suggesting that these were just excess template. Nonetheless, Swati set up cultures to miniprep the following day. She also realized that she had been using a template that was 10x more concentrated than it was supposed to be. She proceeded to set up a fourth attempt at a mutagenic PCR, this time with the correct template concentration, primers for mutating a different cutsite than she had previously been attempting, and a higher annealing temperature.

    Week 2

    It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known. Daily Details
    Daily Details:
    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

    Week 3

    It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known. Daily Details
    Daily Details:
    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

    Week 4

    It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known. Daily Details
    Daily Details:
    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.
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  • << Previous Month
    Next Month >>

    Week 1

    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

    Week 2

    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

    Week 3

    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

    Week 4

    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.
    << Previous Month
    Next Month >>
  • << Previous Month
    Next Month >>

    Week 1

    It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.
    Daily Details:
    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

    Week 2

    It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.
    Daily Details:
    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

    Week 3

    It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.
    Daily Details:
    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

    Week 4

    It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.
    Daily Details:
    Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.
    << Previous Month
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