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

 
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<p>Tushar, Chris, Keith, Susung, John, Jean</p>
 
<p>Tushar, Chris, Keith, Susung, John, Jean</p>
 
<li>Inoculated colonies of TdT variants + Pet28b+</li>
 
<li>Inoculated colonies of TdT variants + Pet28b+</li>
 +
 +
<p>Ben, Susung</p>
 +
<li>Discussed possible designs for the hardware. Marked out design goals as well as limitations.</li>
  
 
<h4>July 15th</h4>
 
<h4>July 15th</h4>
Line 151: Line 154:
 
<li>Did Colony PCR of TdT variants + Pet28b+ colonies</li>
 
<li>Did Colony PCR of TdT variants + Pet28b+ colonies</li>
 
<li>Ran a gel of colony PCR, but results came out negative</li>
 
<li>Ran a gel of colony PCR, but results came out negative</li>
 +
 +
<p>Ben, Susung</p>
 +
<li>First draft of fluidics system using Tygon-tubing and solenoid valves</li>
  
 
<h4>July 17th</h4>
 
<h4>July 17th</h4>
Line 156: Line 162:
 
<li>Planned the immobilization of disulfide modified oligos to mercaptosilane treated glass slides</li>
 
<li>Planned the immobilization of disulfide modified oligos to mercaptosilane treated glass slides</li>
 
<li>Designed a disulfide oligonucleotide for immobilization and ordered 3-mercaptopropyl trimethoxysilane</li>
 
<li>Designed a disulfide oligonucleotide for immobilization and ordered 3-mercaptopropyl trimethoxysilane</li>
 +
 +
<p>Ben, Susung</p>
 +
<li>Design the initial thermocycling system using a series of resistors and switches</li>
 +
<li>Gut a broken PCR machine for thermoelectric coolers</li>
  
 
<h4>July 20th-August 17th</h4>
 
<h4>July 20th-August 17th</h4>
Line 162: Line 172:
 
<li>During this time period, we spent most of our time working on our <a href="https://2015.igem.org/Team:Cooper_Union/Practices">High School Summer STEM Outreach</a>.</li>
 
<li>During this time period, we spent most of our time working on our <a href="https://2015.igem.org/Team:Cooper_Union/Practices">High School Summer STEM Outreach</a>.</li>
 
<li>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 <a href="https://2015.igem.org/Team:Cooper_Union/Collaborations">Genspace iGEM Team</a>.
 
<li>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 <a href="https://2015.igem.org/Team:Cooper_Union/Collaborations">Genspace iGEM Team</a>.
 +
 +
<p>Ben, Susung</p>
 +
<li>Sourced parts</li>
  
 
<h2> AUGUST 2015</h2>
 
<h2> AUGUST 2015</h2>
Line 168: Line 181:
 
<p>Tushar, Chris, Keith, Susung, John, Jean</p>
 
<p>Tushar, Chris, Keith, Susung, John, Jean</p>
 
<li>Set up another double digest of Pet28b+ and the TdT variants with XbaI and BamHI</li>
 
<li>Set up another double digest of Pet28b+ and the TdT variants with XbaI and BamHI</li>
 +
 +
<p>Ben, Susung, Karlin</p>
 +
<li>Initial fluidic system ran into problems</li>
 +
<li>Source parts for peristaltic pump as alternative fluid system</li>
 +
<li>Thermocycling parts arrived and being constructed</li>
 +
<li>Gained access to 3D printer, and began printing peltier case</li>
  
 
<h4>August 18th</h4>
 
<h4>August 18th</h4>
Line 174: Line 193:
 
<li>Set up an overnight ligation of Pet28b+ and TdT variants</li>
 
<li>Set up an overnight ligation of Pet28b+ and TdT variants</li>
 
<li>Set up an RNA ligase reaction of PNK treated 43mer and untreated 52mer</li>
 
<li>Set up an RNA ligase reaction of PNK treated 43mer and untreated 52mer</li>
 +
 +
<p>Ben, Susung, Karlin</p>
 +
<li>3D print complete. First prototype of thermal cycler completed</li>
  
 
<h4>August 19th</h4>
 
<h4>August 19th</h4>
Line 180: Line 202:
 
<li>Ran an agarose gel of the PCR products, results were slightly conclusive; performed transformations of ligations of TdT variants + Pet28b+</li>
 
<li>Ran an agarose gel of the PCR products, results were slightly conclusive; performed transformations of ligations of TdT variants + Pet28b+</li>
 
<li>Ran a polyacrimide gel of RNA ligation, and results were also inconclusive</li>
 
<li>Ran a polyacrimide gel of RNA ligation, and results were also inconclusive</li>
 +
 +
<p>Ben, Tushar</p>
 +
<li>Met with Dac Anh in discussing the potential of moving to microfluidics as an alternative</li>
 +
<li>Discussed advantages, disadvtantages, and feasibility</li>
 +
<li>Concluded that it would be better to work from macro-size and scale down</li>
 +
 +
<p>Ben, Susung, Karlin</p>
 +
<li>Peristaltic pump parts arrive</li>
 +
<li>First pump constructed and tested</li>
 +
  
 
<h4>August 20th</h4>
 
<h4>August 20th</h4>
Line 187: Line 219:
 
<li>Made backup colonies of samples that had TdT del1-27 inserts</li>
 
<li>Made backup colonies of samples that had TdT del1-27 inserts</li>
 
<center><img src="https://static.igem.org/mediawiki/2015/2/22/CooperUnion_Notebook_820.png"/></center>
 
<center><img src="https://static.igem.org/mediawiki/2015/2/22/CooperUnion_Notebook_820.png"/></center>
 +
<center><img src="https://static.igem.org/mediawiki/2015/d/de/CooperUnion_Notebook_8201.png"/></center>
 +
 +
<p>Ben, Susung, Karlin</p>
 +
<li>Concerns raised about thermal cycler</li>
 +
<li>Design a better system and order parts</li>
 +
<li>Begin machining necessary components</li>
  
 
<h4>August 24th</h4>
 
<h4>August 24th</h4>
Line 194: Line 232:
 
<li>Made backup colonies of samples that had TdT del26-143 inserts</li>
 
<li>Made backup colonies of samples that had TdT del26-143 inserts</li>
 
<li>Inoculated previous colonies containing TdT del1-27 inserts</li>
 
<li>Inoculated previous colonies containing TdT del1-27 inserts</li>
 +
<center><img src="https://static.igem.org/mediawiki/2015/1/1f/CooperUnion_Notebook_8241.png"/></center>
 +
 +
<p>Ben, Susung, Karlin</p>
 +
<li>Fried a motorshield</li>
 +
<li>More motors and shields are ordered</li>
  
 
<h4>August 25th</h4>
 
<h4>August 25th</h4>
Line 209: Line 252:
 
<li>Sequencing results verified Pet28b+ plasmids containing the TdT del26-143 inserts with an accuracies of over 98%!</li>
 
<li>Sequencing results verified Pet28b+ plasmids containing the TdT del26-143 inserts with an accuracies of over 98%!</li>
 
<li>Transformed all good TdT variants into Rosetta Protein Purification Cells</li>
 
<li>Transformed all good TdT variants into Rosetta Protein Purification Cells</li>
 +
 +
<p>Ben, Susung, Karlin</p>
 +
<li>New parts arrive</li>
 +
<li>New fluid system tested</li>
 +
<li>New thermal cycler constructed</li>
  
 
<h2>SEPTEMBER 2015</h2>
 
<h2>SEPTEMBER 2015</h2>
Line 221: Line 269:
 
<li>Mini-prepped Pet28b+ with ligated TdT del1-27, and TdT del26-143</li>
 
<li>Mini-prepped Pet28b+ with ligated TdT del1-27, and TdT del26-143</li>
 
<li>Designed and ordered a new reverse ligation oligo for RNA ligation reaction</li>
 
<li>Designed and ordered a new reverse ligation oligo for RNA ligation reaction</li>
 +
 +
<p>Ben, Susung, Karlin</p>
 +
<li>Program Arduino to operate motors and thermal cycler</li>
  
 
<h4>September 8th</h4>
 
<h4>September 8th</h4>
 
<p>Tushar, Chris, Keith, Susung, John, Jean</p>
 
<p>Tushar, Chris, Keith, Susung, John, Jean</p>
 
<li>Set up RNA ligation with new reverse ligation oligo</li>
 
<li>Set up RNA ligation with new reverse ligation oligo</li>
 +
 +
<p>Ben, Susung, Karlin</p>
 +
<li>Extra subsequent pieces are machined and printed</li>
 +
<li>Subsystems are all functional</li>
  
 
<h4>September 18th</h4>
 
<h4>September 18th</h4>
 
<p>Tushar, Chris, Keith, Susung, John, Jean</p>
 
<p>Tushar, Chris, Keith, Susung, John, Jean</p>
 
<li>Ran a gel of RNA ligation from September 8th, results were promising. It seems as if there could be spill over from other gels</li>
 
<li>Ran a gel of RNA ligation from September 8th, results were promising. It seems as if there could be spill over from other gels</li>
−
 
+
<li>View our <a href="https://2015.igem.org/Team:Cooper_Union/Results">results</a> for an in-depth analysis of these RNA Ligase reaction results
−
 
+
<center><img src="https://static.igem.org/mediawiki/2015/0/02/CooperUnion_Notebook_9181.jpeg" width="500px"/></center>
−
 
+
  
  

Latest revision as of 01:31, 19 September 2015

Cooper Union 2015 iGEM



Cooper Union iGEM 2015 Notebook

JUNE 2015

June 3rd - 8th

Tushar, Chris, Keith, Susung, John, Jean

  • 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

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    Tushar, Chris, Keith, Susung, John, Jean

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

    • June 18th

    Tushar, Chris, Keith, Susung, John, Jean

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

    • June 22nd

    Tushar, Chris, Keith, Susung, John, Jean

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

    • June 25th

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    Tushar, Chris, Keith, Susung, John, Jean

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

    • June 29th

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    Tushar, Chris, Keith, Susung, John, Jean

  • Sent in pSB1C3 + TdT variant colonies to be sequenced

    • JULY 2015

    July 1st

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    Tushar, Chris, Keith, Susung, John, Jean

  • Did a transformation of ligations of Pet28b and TdT variants

    • July 6th

    Tushar, Chris, Keith, Susung, John, Jean

  • Performed inoculations of certain colonies

    • July 7th

    Tushar, Chris, Keith, Susung, John, Jean

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

    • July 8th

    Tushar, Chris, Keith, Susung, John, Jean

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

    • July 9th

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    Tushar, Chris, Keith, Susung, John, Jean

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

    • July 14th

    Tushar, Chris, Keith, Susung, John, Jean

  • Inoculated colonies of TdT variants + Pet28b+

    • Ben, Susung

  • Discussed possible designs for the hardware. Marked out design goals as well as limitations.

    • July 15th

    Tushar, Chris, Keith, Susung, John, Jean

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

    • Ben, Susung

  • First draft of fluidics system using Tygon-tubing and solenoid valves

    • July 17th

    Tushar, Chris, Keith, Susung, John, Jean

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

    • Ben, Susung

  • Design the initial thermocycling system using a series of resistors and switches
  • Gut a broken PCR machine for thermoelectric coolers

    • July 20th-August 17th

    Chris, Keith, Susung, John, Jean worked on STEM Outreach

    Tushar worked on glass slide immobalization

  • 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.

    Ben, Susung

  • Sourced parts

    • AUGUST 2015

    August 17th

    Tushar, Chris, Keith, Susung, John, Jean

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

    • Ben, Susung, Karlin

  • Initial fluidic system ran into problems
  • Source parts for peristaltic pump as alternative fluid system
  • Thermocycling parts arrived and being constructed
  • Gained access to 3D printer, and began printing peltier case

    • August 18th

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    • Ben, Susung, Karlin

  • 3D print complete. First prototype of thermal cycler completed

    • August 19th

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    • Ben, Tushar

  • Met with Dac Anh in discussing the potential of moving to microfluidics as an alternative
  • Discussed advantages, disadvtantages, and feasibility
  • Concluded that it would be better to work from macro-size and scale down

    • Ben, Susung, Karlin

  • Peristaltic pump parts arrive
  • First pump constructed and tested

    • August 20th

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    • Ben, Susung, Karlin

  • Concerns raised about thermal cycler
  • Design a better system and order parts
  • Begin machining necessary components

    • August 24th

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    • Ben, Susung, Karlin

  • Fried a motorshield
  • More motors and shields are ordered

    • August 25th

    Tushar, Chris, Keith, Susung, John, Jean

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

    • August 26th

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    • Ben, Susung, Karlin

  • New parts arrive
  • New fluid system tested
  • New thermal cycler constructed

    • SEPTEMBER 2015

    September 2nd

    Tushar, Chris, Keith, Susung, John, Jean

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

    • September 3rd

    Tushar, Chris, Keith, Susung, John, Jean

  • 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

    • Ben, Susung, Karlin

  • Program Arduino to operate motors and thermal cycler

    • September 8th

    Tushar, Chris, Keith, Susung, John, Jean

  • Set up RNA ligation with new reverse ligation oligo

    • Ben, Susung, Karlin

  • Extra subsequent pieces are machined and printed
  • Subsystems are all functional

    • September 18th

    Tushar, Chris, Keith, Susung, John, Jean

  • Ran a gel of RNA ligation from September 8th, results were promising. It seems as if there could be spill over from other gels
  • View our results for an in-depth analysis of these RNA Ligase reaction results