Date/Time | What we plan | Summary of what we did |
Week 1: 4/24-26/2017 |
Monday 4/24/2017 4-6pm |
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- Gathered items needed to be autoclaved
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Tuesday 4/25/2017 12:30-1:00 3:00-5:30 | - Prepare LB broth
- Autoclaved LB broth, pipet tips, and microtubes.
- E.coli Inoculation: Prepare liquid culture with LB nutrient broth and E.coli (previously stored in -80C). Inoculate overnight for 12 hours at 37 degrees with shaking.
- Spectrophotometer testing with dead E.coli
| - Prepared 400mL LB broth
- Autoclaved LB broth, pipet tips, and microtubes.
- E.coli Inoculation: Prepare liquid culture with LB nutrient broth and E.coli (previously stored in -80C). Inoculate overnight for 12 hours at 37 degrees with shaking.
- Spectrophotometer testing with dead E.coli to try to find a correlation between cell concentration and OD600 value (could not find a correlation)
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Wednesday 4/26/2017 10am-5pm | - Prepare different concentration samples of methanol/ethanol. 10% intervals starting from 10% to 60%
- Place E.coli in different samples and perform Spectrophotometer testing to detect the initial and final concentration of E.coli every half an hour
| - Prepared 800mL LB broth
- Prepared LB agar plates and LB/ampicillin agar plates
- Inoculate E.coli
- 20% ethanol survival test for 2 hours (15 min intervals for the first hour and 30 min intervals for the second hour)
- Correlation test for Bacterial Concentration and OD600- used alive E.coli (found no correlation again)
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Week 2: 5/31/2017-6/2/2017 |
Wednesday 5/31/2017 2:00-4:00pm | Plan: - Inoculate E.coli
- Spread glycerol stock E.coli on agar plates
Equipment: Sterile hood, spreader, shaker Reagents: LB broth, agar plates | - Inoculated 6 tubes of E.coli. Started shaking incubator at 3:30pm.
- Revived glycerol stock by plating it onto 4 agar plates. Incubate overnight at 37C.
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Thursday 6/1/2017 1:00-5:00pm
| Plan: - Spread inoculated E.coli on agar plates.
- If there is enough E.coli, conduct survival test in ethanol concentration at 10%, 20%, 30%, and 40%.
Equipment: Sterile Hood, spreader, spectrophotometer Reagents: Ethanol, agar plates, LB broth | - Turn off shaking incubator and placed the tubes in 4C at 10:30am.
- 5 E.coli survival trials at 30% and 40% ethanol concentration (15 min intervals for the first hour and 30 min interval for the remaining two hours).
- Inoculated 4 tubes of E.coli. Start shaking incubator at 5pm
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Friday 6/2/2017 9am-1pm | Plan: - E.coli Survival test at 10% and 20% ethanol concentration.
- Try to find the maximum time at the optimal concentration.
Equipment: Sterile Hood, spreader, spectrophotometer Reagents: Ethanol, LB broth, agar plates | - (2) 5 E.coli survival trials at 10% ethanol concentration.
- First method: using ethanol as blank, get rid of LB broth
- Second method: made alcohol solution using cell culture LB broth rather than water
- Tested for 2 hours. 15 min intervals for the first hour and 30 min intervals for the second hour.
- Learned the procedures for trypan blue staining
- Competent DH5a E.coli cells arrived today
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Week 5: 6/5-9/2017 |
Tuesday 6/6/2017 1:00-5:00pm | Plan: - Inoculate E.coli
- Dead E.coli trypan blue staining under microscope
Equipment: Sterile Hood, spreader, microscope, counting grid Reagents: LB broth, agar plates, trypan blue dye
| - Begin development of ethanol/methanol resistant bacteria: Inoculate E. coli in 1% ethanol or methanol
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Wednesday 6/7/17 9am-5pm | Plan: - E.coli Survival test at 10% and 5% ethanol concentration.
- Inoculate 4 tubes of E.coli
Equipment: Sterile Hood, spreader, shaking incubator, microscope, counting grid Reagents: Ethanol, agar plates, LB broth, trypan blue dye | - Could not distinguish E.coli cells from contaminants on the grid
- Ethanol and methanol resistant bacteria:
- Spread plates for 1% resistance; inoculate 2% resistance from the previous 1% inoculation
- Spread plates for 2% resistance; inoculate 3% resistance from the previous 2%
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Thursday 6/8/17 9am-5pm | Plan: - Inoculate 1 colony each from the 1% ethanol/methanol plates
- Spread plates for 3% resistance; inoculate 4% resistance from the previous 3%
- If dye arrives, practice fluorescence microscope
Equipment: Sterile Hood, spreader, shaking incubator, microscope, counting grid Reagents: Ethanol, agar plates, LB broth, trypan blue dye | - Tried trypan blue staining using new microscope from genetics lab. Was not able to see the E.coli or the counting grids. Abandon using this technique.
- Inoculated E.coli in 4% methanol/ethanol solution for 6 hours. Spread onto agar plates to incubate.
- Spread 3% E.coli on agar plates
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Week 6: 6/19-23/2017 |
Monday 6/19/17 4-5pm | Plan: | - Inoculated 2 tubes of regular LB broth
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Tuesday 6/20/17 9-5pm
Amy and Agnes | Plan: - Prepare E.coli solution
- Prepare working solution for the dyes
- Learn to use the fluorescent microscope from Professor Wenshu. Perform initial testing together.
- Survival test 5% and 10% using the fluorescent microscope
- Inoculate E.coli
| - Test different concentration of E.coli and dye under fluorescent microscope using the hoechst and propidium iodide dyes.
- Gene arrived. Stored in -20
- Inoculated 3 tubes of E.coli
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Wednesday 6/21/17 9-5pm
Amy, Agnes, Julie, Simba | Plan: - Survival test 5% and 10% using the fluorescent microscope
- Inoculate E.coli
Gene arrives: - Prepare plasmid
- Transformation
- Grow overnight on agar plates
| - Tested out fluorescence microscope, varying the variables of dye amount and inoculation dilution
- Cancelled plans for Gas Chromatography, initiated further research for methanol concentration testing
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Thursday 6/22/17 9-5pm | - Survival test 5% and 10% using the fluorescent microscope
- Calibrating the OD600 measurement for DH5a.
- Inoculate one colony of transformed E.coli
| - Tested out fluorescence microscope, varying the variables of dye amount and inoculation dilution
- Postponed OD600 calibration, continued Methanol Concentration testing research
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Week 7: 6/26-30/2017 |
Monday 6/26/17 11am-5pm Agnes and Eric | Plan: - Autoclave and pour agar plates
- Inoculate E.coli
Materials and Instruments: Sterile hood, autoclave | - Poured 40 10 mm agar plates and inoculated 3 tubes of E. coli
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Tuesday 6/27/17 9-5pm Agnes, Amy, Eric | - Fluorescence microscopy: E. coli ethanol survival testing
Materials and Instruments: Sterile hood, fluorescence microscope | - Final Day of E.coli viability testing, increased concentration of dyes and found improved results
- Inoculated 2 tubes of E. coli
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Wednesday 6/28/17 9-5pm Agnes, Amy | - Fluorescence microscopy: E. coli ethanol survival testing
Materials and Instruments: Sterile hood, fluorescence microscope | - Methanol survival testing: at 20 minutes of varying levels of methanol concentration (10%, 20%, 30%, 40%), we looked at the effect on fluorescence in our E. coli samples
- Began methanol clonogenic assay experiment, plated E. coli exposed to varying levels of methanol for 20 minutes (0%, 5%, 10%, 15%, 20%), at 10x dilution
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Thursday 6/29/17 9-5pm | - Fluorescence microscopy: E. coli ethanol survival testing
- Methanol clonogenic assay
Materials and Instruments: Sterile hood, fluorescence microscope | - Began OD600 Calibration, plated dilutions of 200x, 400x, 500x, 800x, and 1000x
- Methanol survival testing continued, varied concentration of dye and exposure to 20% methanol compared to the control
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Friday 6/30/17 9-5pm | - Methanol clonogenic assay
- Fluorescence microscopy: E. coli ethanol survival testing
Materials and Instruments: fluorescence microscope | - Visualized the results of previous day’s plating and updated lab reports
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Week 8: 7/3-7/2017
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Monday 7/3/2017 4-5pm Agnes and Eric | Plan: Materials and Instruments: | |
Tuesday 7/4/17 10-5pm
Julie, Agnes | Plan: - Calibrating OD 600 value
- Inoculate E.coli
Materials and Instruments:
| - Calibration: Plated x200
- Inoculate 2 tubes
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Wednesday 7/5/17 10-5pm
Julie, Simba, Agnes | Plan: - Fluorescence Microscope testing
- Inoculate E.coli
Materials and Instruments: - Sterile room
- Fluorescence Microscope
| - Calibration: Plated x200 and x500
- Inoculate 2 tubes
- Fluorescence microscope: varied time, switched water for PBS
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Thursday 7/6/17 10-5pm
Julie, Agnes | Plan: - Methanol clonogenic assay
- Inoculate E.coli
Materials and Instruments: - Sterile room
- Fluorescence Microscope
| - Calibration: Plated x2000
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Friday 7/7/17 10-11am | Plan: | |
Week 9: 7/10-14/2017Note: Methanol concentration testing will proceed if all chemicals arrive by Tuesday 7/11; Update: Chemicals have not arrived by Tuesday 7/11 |
Monday 7/10/2017 (10)4-5pm
Agnes, Eric | Plan: - Inoculate E.coli
- (Prepare Magenta Sulfite Colorimetric solutions)
Materials and Instruments: | - Inoculate 2 tubes E. coli
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Tuesday 7/11/17 10-5pm | Plan: - Fluorescence testing
- OD600 Calibration
- Inoculate E.coli
Materials and Instruments: - Sterile room
- Fluorescence Microscope
| - Fluorescence: Varied exposure time (1 min, 5 min, 10 min) to methanol at a concentration of 15% methanol; controls: no exposure, 50% methanol at 10 minutes
- OD600 Calibration
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Wednesday 7/12/17 10-5pm | Plan: - Pour ampicillin plates
- Inoculate E.coli
Materials and Instruments: | - Methanol Clonogenic assay using 5% and 15% methanol at different incubation times (5, 10, 15, 20)
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Thursday 7/13/17 10-5pm | Plan: - Methanol clonogenic assay
- Inoculate E.coli
Materials and Instruments: | - Methanol clonogenic assay with 15% and 10% methanol at different time intervals (5, 10, 15, 20)
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Friday 7/14/17 10-5pm | Plan: | - Checked on agar plates and took picture
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Week 10: 7/17-21/2017Note: Magenta Sulfite Colorimetric testing will proceed if all chemicals arrive by Tue 7/18 |
Monday 7/17/2017 3-5pm
Agnes, Eric | Plan: - Inoculate E.coli
- Spread new E.coli on LB and LB/amp
Materials and Instruments: | - Inoculated E.coli
- Plated competent E.coli on agar plates to find suitable plating amount.
- Plated normal E.coli on LB/amp plates to check if the antibiotic works
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Tuesday 7/18/17 10-5pm | Plan: - Transformation
- Methanol clonogenic assay
- Inoculate E.coli
Materials and Instruments: | - Methanol clonogenic assay with different concentrations of methanol 20%-50%
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Wednesday 7/19/17 10-5pm | Plan: - Prepare Magenta Sulfite Colorimetric solutions (must be autoclaved)
- Methanol clonogenic assay
- Inoculate E.coli
Materials and Instruments: | - Clonogenic assay with different concentrations of ethanol. 10%-40%
- Filming
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Thursday 7/20/17 10-5pm | Plan: - Magenta Sulfite Colorimetric testing
- Inoculate E.coli
Materials and Instruments: | - Filming
- Fluorescent Microscopy with 30% ethanol and 50% methanol
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Friday 7/21/17 10-115pm | Note: Due to the fact that the chemicals for the Magenta Sulfite Colorimetric testing will be arriving today, we will only go to lab in the morning | - Took the plates out of the incubator. Took pictures
- Filmed scenes for video
- Chemicals arrived!
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Week 11: 7/24-28/2017 |
Monday 7/24/2017 1:30-5pm
Agnes, Eric | Plan: - Inoculate E.coli
- Transformation
Materials and Instruments: | - Inoculated 5 tubes of transformed E.coli
- Prepared chemical solutions
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Tuesday 7/25/17 1-5pm | Plan: - Magenta Sulfite Colorimetric testing
- Inoculate E.coli
Materials and Instruments: - Sterile room, Microplate reader
| - Prepared chemical solutions
- Inoculated 2 tubes of transformed E.coli
- Reviewed Interlab protocols. Just found out that our school’s plate reader cannot measure fluorescence
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Wednesday 7/26/17 10-5pm | Plan: - Magenta Sulfite Colorimetric testing
- Methanol clonogenic assay
- Inoculate E.coli
Materials and Instruments: | - Magenta Sulfite Colorimetric
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Thursday 7/27/17 10-5pm | Plan: - Magenta Sulfite Colorimetric testing
- Inoculate E.coli
Materials and Instruments: | - Magenta Sulfite Colorimetric
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Week 11: 7/31-8/4/2017We will NOT be using the lab this week. Lab work will be moved to Fudan University in order to use their microplate reader for the Interlab. Lab work will continue the following week. |
Week 11: 8/7-8/11/2017We will not be using the lab this week. Lab work will be moved to Fudan University in order to use their microplate reader for the Interlab. Lab work will continue the following week. |
Week 11: 8/14-18/2017 |
Monday 8/14/20171:30-5pm | Plan: - Inoculate E.coli
- Transformation
Materials and Instruments: |
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Tuesday 8/15/1710-5pm | Plan: - Magenta Sulfite Colorimetric testing
- Inoculate E.coli
Materials and Instruments: |
Same as planed |
Wednesday8/16/1710-5pm | Plan: - Magenta Sulfite Colorimetric testing
- Inoculate E.coli
Materials and Instruments: | Same as planned |
Thursday 8/17/1710-5pm | Plan: - Magenta Sulfite Colorimetric testing
Materials and Instruments:
| Same as planned |
