Difference between revisions of "Team:Stony Brook/Notebook"

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<p> Document the dates you worked on your project.</p>
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<h1> Stony Brook iGEM Lab Notebook </h1>
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<h5>What should this page have?</h5>
 
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<ul>
 
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<li>Chronological notes of what your team is doing.</li>
 
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<li> Brief descriptions of daily important events.</li>
 
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<li>Pictures of your progress. </li>
 
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<li>Mention who participated in what task.</li>
 
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<h5>Inspiration</h5>
 
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<p>You can see what others teams have done to organize their notes:</p>
 
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<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
 
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<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
 
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<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
 
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<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
 
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Revision as of 20:01, 19 July 2016

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Stony Brook iGEM Lab Notebook

Week 1 (6/27 - 7/2)

6/27

Cancer Group
  • PCR of 3xHA-TDGF1 construct. 12 cycles
  • Gel of PCR Product → PCR was a failure. Band was 300bp. Should have been 750bp
  • Second PCR of 3xHA-TDGF1 construct. 25 cycles instead of 12 → left overnight
Vaccine Group
  • Things
Interlab Study
  • Measured LUDOX and H2O for plate reader
  • Transformation of positive and negative controls, test devices 1-3

6/28

Cancer Group
  • Ran gel on PCR of yesterday's construct → Gel worked
  • Gel purification of PCR Product → low concentration of DNA found during nanodrop
  • PCR the construct using a revised method
  • 2:54pm → ran the gel on the PCR product
Vaccine Group
  • Things
Interlab Study
  • Heat and "SB" streaking seems not to have worked
  • No visible red colonies on plate

6/29

Cancer Group
  • PCR Not working well → keep trying new methods with different annealing temperatures
  • Tried 65 and 67 degrees C
Vaccine Group
  • Things
Interlab Study
  • FITC started
  • Cutting re-inoculated
  • Ordered new LUDOX
Other
  • College of Arts and Sciences Pre College Summer Institute students stopped by lab
  • Spoke to them about information on synthetic biology and iGEM

6/30

Cancer Group
  • PCR worked → Test 5 → changed annealing temperature to 66 degrees C + increased the denaturing time
  • Nanodropped DNA
Vaccine Group
  • Things

7/1

Cancer Group
  • Digestion of TDGF-1 construct and YFP352GAP vector with two restriction enzymes
  • Will ligate later
TDGF-1 Construct (Phire PCR 128.2 ng/mL) YEP352GAP Vector (343.2 ng/mL)
Total Reaction Volume 50 uL 50 uL
Nuclease Free Water 27.4 uL 37.2 uL
10x Cutsmart Buffer 5 uL 5uL
xho1 1 uL 1 uL
Pvu II 1 uL 1 uL
DNA 15.6 uL 5.8 uL
Vaccine Group
  • Things

7/2

Cancer Group
  • Running yesterday's digestion on gel

Week 2 (7/5-7/8)

7/5

Cancer Group
  • Ran the PCR'ed construct on a gel with Phire Polymerase → It didn't work
Vaccine Group
  • Things

7/6

Cancer group
  • Ran the construct with PCR on gel → it worked
  • Tasnia stabbed our gel
  • Interlab study continues
Vaccine Group
  • Things

7/7

Cancer group
  • Nanodrops were pretty low (84-120 ng/nl)
  • 11:46am PCR'ed the highest nanodrop result in tube #1 → PCR successful
  • 2:33pm → attempt to digest with remaining non-PCR product → removed from incubator at 5:00pm
  • Made a gel → ran PCR
Vaccine Group
  • Things

Week 3 (7/11-7//15)

7/11

Cancer group
  • Ran PCR on the construct (5x) → gel did not work
  • PCR'ed 84.2 ng/ml construct
  • 5 replicates made using Phire
  • Thermocycler Program

Thermocycler Program
Step Temperature (Degrees C) Duration (seconds)
1 98 30
2 98 20
3 66 5
4 72 30
5 72 60
6 4 Hold
After step 4, return to step 2. Repeat steps 2-4 thirty-five times before continuing to step 5 and on.
Water 19.5 uL
Phire Polymerase 25 uL
Template 0.5 uL
Forward Primer 2.5 uL
Reverse Primer 2.5 uL
  • Restreaking of YEP352GAP transformed cells for miniprep
  • Restreaking placed in incubator at 12am
Vaccine Group
  • Stuff & Things

7/12

Cancer group
  • Made gel for electrophoresis of PCR construct → it didn't work
  • Thermocycler Program: Same as PCR from 7/11

Q5 Rx Buffer 10 uL
10nM dNTPs 1 uL
10 uM Forward Primer 2.5 uL
10 uM Reverse Primer 2.5 uL
Template (Q5 PCR Pw.1 for 6/30/16) 0.5 uL
Q5 Polymerase 2.5 uL
Water 33 uL
  • LB liquid culture (3ml with 3ul of ampicillin)
  • Loaded 10ul Ladder DNA.
  • Loaded 1ul dye + 5ul PCR product
  • Ran gel at 115V
  • Checked gel ladder → faint, no band for the PCR product
  • Made new gel, loaded with the same amount of reagents as before → ran at 90V → No ladder band
  • Ran it again at 115V → visible ladder, no PCR band
  • Preparing a gel to run ladder and construct that previously showed a strong band → used this to diagnose a problem with gel setup. Ran TDGF1 152.9 Phire Child. Lane 1 is ladder, Lane 2 is that construct (10ul) with 2ul of dye
  • Setup new PCR using the 6/30/16 PCR construct
  1. 98°C → 30sec
  2. 98°C → 20sec
  3. 66°C → 5sec
  4. 72°C → 30sec
  5. 72°C → 1min
  6. 4°C → hold
Vaccine Group
  • Stuff & Things

7/13

Cancer group
  • Ran 5ul of PCR with ladder, cancer construct and vaccine construct → to diagnose problem with gel
  • Miniprep of Dean vector (3ml LB culture)
  • Purification of PCR product → nanodropped 16.4ug/ul
  • Nanodrop of:
  1. Yellow 7/13 construct NEB purified → 16.4ng/ml
  2. Blue gel purification → 48.7ng/ul
  3. Pink PCR purification 7/13 → 102.2ng/ul
  4. 7/13 -RK Vector 1 → 48.2ng/ul
  5. 7/13 -RK Vector 2 → 96.3ng/ul
Vaccine Group
  • Things

7/14

Cancer group
  • Things
Vaccine Group
Contents of well Amount (ul)
Ladder 10
AD 5
AO 5
AM 5
AN 5
Gal-Sunil Added 1ul loading dye
Con-Sunil 5
  • Brian and Sunil's Gal promoters worked but both constructs failed
Biobrick ideas
  1. Smelly yeast → optimized from e. coli
  2. Twist on smelly e. coli on e. coli → regulated with quorum sensing
  3. OmpA/EnvZ recognize osmolarity
  4. Genetic Circuit
  5. Shine yellow light → Banana smell. Shine green light → wintergreen
  6. TetR operator system genetic circuit
  7. Have cell die at certain wavelength → killswitch

7/15

Cancer group
  • Ran a gel on PCR products →
Vaccine group
  • Jon and Sunil retrying construct
  • Jon used phire at 50°C
  • Sunil used Q5. Extension at 72°C for 30 seconds
Polymerase Primers (ul) Template (ul) H2O (ul)
Phire (25ul) 2.5 Reverse Construct 2.5 Forward Stitch 1.5 19.5
Q5 (25ul) 2.5 Reverse Construct 2.5 Forward Stitch 1.5 19.5