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

Line 89: Line 89:
 
<li>Ran PCR products on a gel</li>
 
<li>Ran PCR products on a gel</li>
 
<li>Made backup colonies for cells that had vector + insert</li>
 
<li>Made backup colonies for cells that had vector + insert</li>
 +
<left><img src="https://static.igem.org/mediawiki/2015/f/fd/CooperUnion_Notebook_6291.jpeg" width="500px"/></left>
  
 
<h4>June 30th</h4>
 
<h4>June 30th</h4>

Revision as of 06:13, 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 following sequences were designed: TdT delta1-27 (BBa_K1746000), TdT delta26-143 (BBa_K1746001), and TdT GIP 213-215 subAAA (BBa_K1746002).

    • 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.

    AUGUST 2015

    August 17th

  • Set up another double digest of Pet28b+ and the TdT variants with XbaI and BamHI

    • August 18th

  • Performed a PCR cleanup of the double digests the Pet28b+ and TdT variants
  • Set up an overnight ligation of Pet28b+ and TdT variants
  • Set up an RNA ligase reaction of PNK treated 43mer and untreated 52mer

    • August 19th

  • To verify if ligation worked, instead of transforming immediately, performed PCR of ligation product
  • Ran an agarose gel of the PCR products, results were slightly conclusive; performed transformations of ligations of TdT variants + Pet28b+
  • Ran a polyacrimide gel of RNA ligation, and results were also inconclusive

    • August 20th

  • Performed colony PCR of 5 colonies from each plate (5 from TdT del1-27 + Pet28b+; 5 from TdT del26-143 + Pet28b+; 5 from TdT GIP subAAA + Pet28b+)
  • Results were positive! Indicated TdT del1-27 inserts in Pet28+
  • Made backup colonies of samples that had TdT del1-27 inserts

    • August 24th

  • Performed more colony PCR of the same plates from August 20th, to verify if any other colonies had inserts
  • Ran gel of colony PCR and verified cells with inserts containing TdT del26-143
  • Made backup colonies of samples that had TdT del26-143 inserts
  • Inoculated previous colonies containing TdT del1-27 inserts

    • August 25th

  • Mini-prepped inoculated TdT del1-27 colonies and sent plasmid to be sequenced
  • Inoculated previous colonies containing TdT del26-143 inserts

    • August 26th

  • Sequencing results verified Pet28b+ plasmids containing the TdT del1-27 inserts with an accuracies of over 97%!
  • Prepared and sent previous TdT del26-143 samples for sequencing

    • August 27th

  • Sequencing results verified Pet28b+ plasmids containing the TdT del26-143 inserts with an accuracies of over 98%!
  • Transformed all good TdT variants into Rosetta Protein Purification Cells

    • SEPTEMBER 2015

    September 2nd

  • Inoculated Rosetta cells containing Pet28b+ with ligated TdT del1-27, and TdT del26-143

    • September 3rd

  • Used some inoculate to make backup colonies of Pet28b+ with ligated TdT del1-27, and TdT del26-143
  • Mini-prepped Pet28b+ with ligated TdT del1-27, and TdT del26-143
  • Designed and ordered a new reverse ligation oligo for RNA ligation reaction

    • September 8th

  • Set up RNA ligation with new reverse ligation oligo