Team:Minnesota/Notebook
From 2009.igem.org
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+ | <h1>The Experiments</h1> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h1>Results</h1> | ||
+ | |||
+ | |||
+ | |||
+ | <h1>Protocols: Standard techniques that we used in the wet lab</h1> | ||
+ | <h3>Bacterial Culture</h3> | ||
+ | <h4>Sterile Technique</h4> | ||
+ | <ol> | ||
+ | <li>Always work around a flame or in the hood</li> | ||
+ | <li>Flame the mouth and cap of any bottle, flask or tube upon uncapping and recapping</li> | ||
+ | <li>Sterilize metal instruments between uses by dipping in 100% ethanol and flaming</li> | ||
+ | </ol> | ||
+ | <br /> | ||
+ | <h4>Bacterial Culture Maintenance</h4> | ||
+ | Culture cells: | ||
+ | <ol> | ||
+ | <li>At 36 degrees Celsius</li> | ||
+ | <li>Shaking at 220 rpm</li> | ||
+ | <li>At 10% total flask/tube volume</li> | ||
+ | <li>In mid-log phase(0.1 < OD600 <= 0.4) (with OD600 = 1 ->8.8x10<sup>8</sup>cell/ml) | ||
+ | </ol> | ||
+ | <br /> | ||
+ | <h4>Bacterial Culture For Gene Expression Experiments</h4> | ||
+ | <ol> | ||
+ | <li>Pick and individual colony from a plate and inoculate 2ml LB + amp media</li> | ||
+ | <li>Incubate overnight at 37 C, shaking at 220 rpm</li> | ||
+ | <li>Inoculate fresh media with overnight culture such that new culture has 2.5% inoculum; this is the secondary culture</li> | ||
+ | <li>Incubate at 37 C shaking at 220 rpm until OD600 = 0.4 (~2 hrs)</li> | ||
+ | <li>Inoculate 4 ml LB + amp + inducer (aTc or IPTG) with 100ul secondary culture</li> | ||
+ | <li>Continue cultures as described above in "bacterial culture maintenance" for 9 hrs</li> | ||
+ | <li>Isolate cell samples from cultures at 3, 6, and 9 hour time points</li> | ||
+ | <li>Remove 100ul sample aliquots from cultures</li> | ||
+ | <li>Pellet samples at 5K rpm for 5 minutes</li> | ||
+ | <li>Remove supernatant</li> | ||
+ | <li>Wash cells with 1 ml chilled 1xPBS, pH 7.6</li> | ||
+ | <li>Resuspend cells by vortexing</li> | ||
+ | <li>Re-pellet cells at 5K rpm for 5 minutes</li> | ||
+ | <li>Remove supernatant</li> | ||
+ | <li> Fix cells; resuspend cells in 1 ml 4% PFA (in PBS)</li> | ||
+ | <li>Incubate at RT for 30 minutes</li> | ||
+ | <li>Pellet cells at 5K rpm for 5 minutes</li> | ||
+ | <li>Remove supernatant</li> | ||
+ | <li>Resuspend cells in 1 ml 1xPBS</li> | ||
+ | <li> Store samples at 4 C until analysis by flow cytometry</li> | ||
+ | </ol> | ||
+ | <br /> | ||
+ | <h4>Transformation of Chemically Competent Cells</h4> | ||
+ | <ol> | ||
+ | <li>Thaw cells and incubate transformant DNA in ice(~15 minutes)</li> | ||
+ | <li>Combine 50 ul cells with ~3uL DNA and mix gently</li> | ||
+ | <li>Incubate samples on ice for 15 minutes</li> | ||
+ | <li>Heat shock cells in 42C water bath of 50 seconds</li> | ||
+ | <li>Incubate samples on ice for 5 minutes</li> | ||
+ | <li>Recover cells in 0.5 ml SOC media, shaking at 37C for 1 hour at 220 rpm</li> | ||
+ | <li>Transfer cells to a 2 ml microfuge tube</li> | ||
+ | <li>Spin cells down at 6K rpm for 2 minutes</li> | ||
+ | <li>Remove all but ~100uL supernatant media</li> | ||
+ | <li>Resuspend cells gently in remaining media</li> | ||
+ | <li>Plate cells on LB + ab plates</li> | ||
+ | <li>Incubate plates overnight at 37C</li> | ||
+ | </ol> | ||
+ | <br /> | ||
+ | <br /> | ||
+ | <br /> | ||
+ | <h3>DNA Work</h3> | ||
+ | <br /> | ||
+ | <h4>Plasmid Prep from cultures (using QIAprep Spin Miniprep Kit)</h4> | ||
+ | <ol> | ||
+ | <li>Pick and individual colony from a plate and inoculate 2ml LB + ab media</li> | ||
+ | <li>Incubate culture overnight at 37C</li> | ||
+ | <li>Transfer culture to 2 ml microfuge tube</li> | ||
+ | <li>Spin cells down at 13K rpm for 2 min at RT and remove supernatant</li> | ||
+ | <li>Resuspend cells in 250 ul Buffer P1 (stored at 4 C)</li> | ||
+ | <li>Add 250 ul Buffer P2 and mix thoroughly by inverting-- the solution should turn blue</li> | ||
+ | <li>Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting-- the solution should turn colorless</li> | ||
+ | <li>Centrifuge sample at 13K rpm for 10 minutes</li> | ||
+ | <li>Transfer supernatant to a fresh QIAprep spin column, leaving cell debris pellet behind</li> | ||
+ | <li>Centrifugre supernatant into column at 13K rpm for 1 minute</li> | ||
+ | <li>Remove the flowthrough</li> | ||
+ | <li>Wash column with 0.5 ml Buffer PB; apply to column and spin through at 13K rpm for 1 minute</li> | ||
+ | <li>Remove the flowthrough</li> | ||
+ | <li>Wash column with 0.75 ml Buffer PE; apply to column and spin through at 13K rpm for 1 minute</li> | ||
+ | <li>Remove the flowthrough</li> | ||
+ | <li>Spin out residual liquid at 13K rpm for 1 minute</li> | ||
+ | <li>Place column in a fresh 1.5 ml microfuge tube</li> | ||
+ | <li>Elute DNA; apply 40 ul Buffer EB to column, incubate at room temperature for 2 minutes and spin out of column at 13K rpm for one minute</li> | ||
+ | </ol> | ||
+ | <br /> | ||
+ | <h4>DNA quantification</h4> | ||
+ | <ol> | ||
+ | <li>Diluite DNA as appropriate in water (1<= DF <=1/100) to a total volume of 50 ul</li> | ||
+ | <li>Similarly dilute blank DNA buffer solution with water to a total volume of 50 ul</li> | ||
+ | <li>Read absobance of blank and DNA sample at lambda = 260 and 280</li> | ||
+ | <li>Calculate [DNA]; [DNA](ng/ul) = DF*A260*50</li> | ||
+ | <li>Determine sample purity; pure DNA A260/A280 = 1.8</li> | ||
+ | </ol> | ||
+ | <br /> | ||
+ | |||
<h2>Our Google Calendar</h2> | <h2>Our Google Calendar</h2> | ||
<iframe src="http://www.google.com/calendar/embed?height=600&wkst=1&bgcolor=%23FFFFFF&src=2o8bfumseou1ivsgaknp0kfh7c%40group.calendar.google.com&color=%230D7813&ctz=America%2FChicago" style=" border-width:0 " width="800" height="600" frameborder="0" scrolling="no"></iframe> | <iframe src="http://www.google.com/calendar/embed?height=600&wkst=1&bgcolor=%23FFFFFF&src=2o8bfumseou1ivsgaknp0kfh7c%40group.calendar.google.com&color=%230D7813&ctz=America%2FChicago" style=" border-width:0 " width="800" height="600" frameborder="0" scrolling="no"></iframe> |
Revision as of 16:54, 16 July 2009
Home | The Team | The Project | Submitted Parts | Modeling | SynBioSS Designer | Parts Characterization | Experiments |
---|
The Experiments
Results
Protocols: Standard techniques that we used in the wet lab
Bacterial Culture
Sterile Technique
- Always work around a flame or in the hood
- Flame the mouth and cap of any bottle, flask or tube upon uncapping and recapping
- Sterilize metal instruments between uses by dipping in 100% ethanol and flaming
Bacterial Culture Maintenance
Culture cells:- At 36 degrees Celsius
- Shaking at 220 rpm
- At 10% total flask/tube volume
- In mid-log phase(0.1 < OD600 <= 0.4) (with OD600 = 1 ->8.8x108cell/ml)
Bacterial Culture For Gene Expression Experiments
- Pick and individual colony from a plate and inoculate 2ml LB + amp media
- Incubate overnight at 37 C, shaking at 220 rpm
- Inoculate fresh media with overnight culture such that new culture has 2.5% inoculum; this is the secondary culture
- Incubate at 37 C shaking at 220 rpm until OD600 = 0.4 (~2 hrs)
- Inoculate 4 ml LB + amp + inducer (aTc or IPTG) with 100ul secondary culture
- Continue cultures as described above in "bacterial culture maintenance" for 9 hrs
- Isolate cell samples from cultures at 3, 6, and 9 hour time points
- Remove 100ul sample aliquots from cultures
- Pellet samples at 5K rpm for 5 minutes
- Remove supernatant
- Wash cells with 1 ml chilled 1xPBS, pH 7.6
- Resuspend cells by vortexing
- Re-pellet cells at 5K rpm for 5 minutes
- Remove supernatant
- Fix cells; resuspend cells in 1 ml 4% PFA (in PBS)
- Incubate at RT for 30 minutes
- Pellet cells at 5K rpm for 5 minutes
- Remove supernatant
- Resuspend cells in 1 ml 1xPBS
- Store samples at 4 C until analysis by flow cytometry
Transformation of Chemically Competent Cells
- Thaw cells and incubate transformant DNA in ice(~15 minutes)
- Combine 50 ul cells with ~3uL DNA and mix gently
- Incubate samples on ice for 15 minutes
- Heat shock cells in 42C water bath of 50 seconds
- Incubate samples on ice for 5 minutes
- Recover cells in 0.5 ml SOC media, shaking at 37C for 1 hour at 220 rpm
- Transfer cells to a 2 ml microfuge tube
- Spin cells down at 6K rpm for 2 minutes
- Remove all but ~100uL supernatant media
- Resuspend cells gently in remaining media
- Plate cells on LB + ab plates
- Incubate plates overnight at 37C
DNA Work
Plasmid Prep from cultures (using QIAprep Spin Miniprep Kit)
- Pick and individual colony from a plate and inoculate 2ml LB + ab media
- Incubate culture overnight at 37C
- Transfer culture to 2 ml microfuge tube
- Spin cells down at 13K rpm for 2 min at RT and remove supernatant
- Resuspend cells in 250 ul Buffer P1 (stored at 4 C)
- Add 250 ul Buffer P2 and mix thoroughly by inverting-- the solution should turn blue
- Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting-- the solution should turn colorless
- Centrifuge sample at 13K rpm for 10 minutes
- Transfer supernatant to a fresh QIAprep spin column, leaving cell debris pellet behind
- Centrifugre supernatant into column at 13K rpm for 1 minute
- Remove the flowthrough
- Wash column with 0.5 ml Buffer PB; apply to column and spin through at 13K rpm for 1 minute
- Remove the flowthrough
- Wash column with 0.75 ml Buffer PE; apply to column and spin through at 13K rpm for 1 minute
- Remove the flowthrough
- Spin out residual liquid at 13K rpm for 1 minute
- Place column in a fresh 1.5 ml microfuge tube
- Elute DNA; apply 40 ul Buffer EB to column, incubate at room temperature for 2 minutes and spin out of column at 13K rpm for one minute
DNA quantification
- Diluite DNA as appropriate in water (1<= DF <=1/100) to a total volume of 50 ul
- Similarly dilute blank DNA buffer solution with water to a total volume of 50 ul
- Read absobance of blank and DNA sample at lambda = 260 and 280
- Calculate [DNA]; [DNA](ng/ul) = DF*A260*50
- Determine sample purity; pure DNA A260/A280 = 1.8