Difference between revisions of "Team:Cooper Union/Notebook"

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Revision as of 00:36, 18 September 2015

Cooper Union 2015 iGEM



Cooper Union iGEM 2015 Notebook

JUNE 2015

June 3rd - 8th

  • We designed various truncated and modified TdT sequences using the SnapGene viewer. The sequences were derived from the results documented in a paper by Repasky et. all detailing the Mutational Analysis of Terminal Deoxynucleotidyltransferase-mediated N-Nucleotide Addition in V(D)J Recombination.
  • The sequences can be found in the following google drive folder: https://drive.google.com/drive/folders/0ByXS6yhMrkJ7flpzTjF2cXFLTFEzUHBlUC1CTU9pd2FObmZyc0ozNTBGSnhxTnk3VGZMZzQ/0B713y2SjhYhifklQeXlvOTFSVkRTakVKREZ6dW0xTWRIVVVoWlBiM0ZZbllCdWpPTzBpVTQ/0B713y2SjhYhifldYeVRlRkZ5ZWJlRi1YUmlxOXFvZEFaa2ZBY3VMLWFLNHQyWmwzNmlPQk0/0B713y2SjhYhiflF5MU5nMnh4bExsS3h2dkx6S2JEcWxPZWRCRVNMX1lGZkp1TTc1alZad1U

    • June 16th

  • We practiced making 1% agarose gels. We immobilized DNA to Dynabeads (following the Dynabead Prep Protocol) and then ran experiments with TdT using regular dATPs and Cleanamp dATPs (following the Dynabead Cleanamp Protocol). We ran a gel of the experiments but saw no results. It is proposed that DNA was lost when removing DNA from Dynabeads.

    • June 17th

  • Prepared more Dynabeads with immobalized DNA (following the Dynabead Prep Protocol)
  • Prepared LB Broth
  • Prepared more 1% agarose gels

    • June 18th

  • Ran gels of Cleanamp Dynabead Experiment, but saw no results.

    • June 22nd

  • To test our polyacrimide gels (they were kind of old) we ran another polyacrimide gel with regular DNA sequences

    • June 25th

  • Received the TdT del1-27. del26-143, and GIP subAAA variants!
  • Did a double digest of TdT variants and pSB1C3 ADD PROTOCOL
  • PCR purified restriction enzyme digest products and then performed a ligation with T4 DNA Ligase ADD PROTOCOL

    • June 26th

  • Performed a Transformation of TdT variants cloned into pSB1C3 with NEB5alpha cells

    • June 29th

  • Made two 1% agarose gels
  • Observed that colonies grew on our transformation plates
  • Did a colony PCR of multiple colonies
  • Ran PCR products on a gel
  • Made backup colonies for cells that had vector + insert

    • June 30th

  • Sent in pSB1C3 + TdT variant colonies to be sequenced

    • JULY 2015

    July 1st

  • Followed Double Digest Protocol for Pet28b+ with BamHI and XbaI; this includes the double digest of Pet28b+ and TdT del 1-27. del 26-143, and GIP sub AAA TdT mutants
  • Did PCR purification - Really low concentration and bad graph on nanodrop
  • Was recommended to use less EB buffer in the future>
  • Reran digestion of Pet28b+
  • Did PCR purification with less EB buffer
  • Performed a ligation of Pet28b+ and TdT variants using T4 DNA Ligase

    • July 2nd

  • Did a transformation of ligations of Pet28b+ and TdT variants

    • July 6th

  • Did an innoculation of Pet28b+ and TdT variants

    • July 7th

  • Performed a miniprep of innoculated TdT + Pet28b+ plasmids
  • Nano-dropped samples and prepared them to be sequenced

    • July 8th

  • Sent samples from yesterday to be sequenced
    • should this page have?
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    • Brief descriptions of daily important events.
    • Pictures of your progress.
    • Mention who participated in what task.

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