Difference between revisions of "Team:Stony Brook/Notebook"
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| − | < | + | <th>TDGF-1 Construct (Phire PCR 128.2 ng/mL)</th> |
| − | < | + | <th>YEP352GAP Vector (343.2 ng/mL)</th> |
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<td colspan=3 align=center>Thermocycler Program</td> | <td colspan=3 align=center>Thermocycler Program</td> | ||
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| − | < | + | <th>Step</th> |
| − | < | + | <th>Temperature (Degrees C)</th> |
| − | < | + | <th>Duration (seconds)</th> |
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<tr> | <tr> | ||
Revision as of 19:11, 15 July 2016
Document the dates you worked on your project.
What should this page have?
- Chronological notes of what your team is doing.
- Brief descriptions of daily important events.
- Pictures of your progress.
- Mention who participated in what task.
Inspiration
You can see what others teams have done to organize their notes:
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
- 98°C → 30sec
- 98°C → 20sec
- 66°C → 5sec
- 72°C → 30sec
- 72°C → 1min
- 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:
- Yellow 7/13 construct NEB purified → 16.4ng/ml
- Blue gel purification → 48.7ng/ul
- Pink PCR purification 7/13 → 102.2ng/ul
- 7/13 -RK Vector 1 → 48.2ng/ul
- 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
- Smelly yeast → optimized from e. coli
- Twist on smelly e. coli on e. coli → regulated with quorum sensing
- OmpA/EnvZ recognize osmolarity
- Genetic Circuit
- Shine yellow light → Banana smell. Shine green light → wintergreen
- TetR operator system genetic circuit
- Have cell die at certain wavelength → killswitch
7/15
Cancer group
- Stuff & Things
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 |
