Team:Cooper Union/Notebook



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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

  • Performed inoculations of certain colonies

    • July 7h

  • Mini prepped inoculations from previous night
  • Nanodrop of colonies indicated good results and sent the samples to be sequenced

    • July 8th

  • Sequencing results indicated no priming for samples, and therefore ligation did not work or the DNA was lost in some process afterwards

    • July 9th

  • Re-ran double digests of Pet28b+ and TdT variants
  • Performed a gel purification of the digests and set up an overnight ligation of TdT variants + Pet28b+

    • July 10th

  • Made fresh LB Agar + Kanamycin plates
  • Transformed ligations from July 9th onto fresh Kan-plates

    • July 14th

  • Inoculated colonies of TdT variants + Pet28b+

    • July 15th

  • Did Colony PCR of TdT variants + Pet28b+ colonies
  • Ran a gel of colony PCR, but results came out negative

    • July 17th

  • Planned the immobilization of disulfide modified oligos to mercaptosilane treated glass slides
  • Designed a disulfide oligonucleotide for immobilization and ordered 3-mercaptopropyl trimethoxysilane

    • July 20th-August 17th

  • During this time period, we spent most of our time working on our High School Summer STEM Outreach.
  • We also spent our time perfecting our technique for creating mercaptosilane treated glass slides, and immobilizing disulfide modified DNA to them. This process involved a collaboration with the Genspace iGEM Team.
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