Difference between revisions of "Team:Virginia/Experiments"

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<div style = 'padding-right: 90px; padding-left: 90px; text-indent: 50px;line-height: 25px;' >
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<body>
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<div class="bg-primary">
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<section>
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<div class="col-lg-16">
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<center>
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            <img src="https://static.igem.org/mediawiki/2016/e/e4/T--Imperial_College--ProtoBanner.png" height="450"/>
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          </center>
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</div>
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<div class="col-lg-10 col-centered">
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<p>On this page, the ecolibrium team would like to share with you the protocols that we have been using over the summer. This library of protocols has been developed alongside our supervisors for the purpose of this study. Here you can find the exact methods we use to generate our data and results. We share them in the interest of reproducibility.<br><br></p>
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<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
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            <div class="panel panel-default">
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                <div class="panel-heading" role="tab" id="P1">
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                    <h4 class="panel-title">
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                    <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P1-collapse" aria-expanded="false" aria-controls="P1-collapse">
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<div>
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                    <div class="col-md-11">Chemical Transformation</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
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</div>
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                    </a>
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                    </h4>
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                </div>
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                <div id="P1-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P1">
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                    <div class="panel-body">
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<p>
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<b>Materials:</b><br>
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LB broth<br>
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Ice<br>
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Selection plates<br>
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<br>
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<b>Methods:</b><br>
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<ol style="font-size:16px;">
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<li>Thaw 50µL competent E. coli cells on ice for 10 minutes<br></li>
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<li>Add:
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<ul style="font-size:16px;">
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<li>5-10 µl DNA from a ligation reaction mix or </li>
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<li>10-100ng DNA of a known plasmid </li>
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</ul>
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</li>
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<li>Carefully flick the tube 4-5 times to mix cells and DNA. <b>Do not vortex.</b></li>
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<li>Place the mixture on ice for 30 minutes. <b>Do not mix.</b></li>
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<li>Heat shock at exactly 42°C for exactly 30 seconds. <b>Do not mix.</b></li>
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<li>Place on ice for 5 minutes. <b>Do not mix.</b></li>
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<li>Pipette 950 µl of room temperature SOC or LB media into the mixture.</li>
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<li>Incubate at 37°C and 200-250 rpm for 60 minutes.</li>
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<li>Mix the cells thoroughly by flicking the tube and inverting.</li>
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<li>Spread:
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<ul style="font-size:16px;">
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<li>For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
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<ol style="font-size:16px;">
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<li>Pellet cells at 8000rpm for 3 minutes</li>
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<li>Remove and dispense 600 µL of supernatant </li>
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<li>Re-suspend cells by light vortexing</li>
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<li>Plate resuspended cells as above</li>
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</ol></li>
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<li>For known plasmid: 10 & 100 µL of each transformation reaction onto a selection plate. For the rest of 890 µL:
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<ol style="font-size:16px;">
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<li>Pellet cells at 8000rpm for 3 minutes</li>
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<li>Remove and dispense 600 µL of supernatant </li>
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<li>Re-suspend cells by light vortexing</li>
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<li>Plate resuspended cells as above</li>
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</ol></li>
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</ul>
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<li>Incubate overnight at 37°C with plates upside down.</li>
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</ol>
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</p>
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    </div>
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  </div>
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</div>
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<div class="some-padding"></div>
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<div class="some-padding"></div>
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<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
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            <div class="panel panel-default">
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                <div class="panel-heading" role="tab" id="P2">
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                    <h4 class="panel-title">
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                    <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P2-collapse" aria-expanded="false" aria-controls="P2-collapse">
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<div>
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                    <div class="col-md-11">Growing Overnight Cultures</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
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</div>
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                    </a>
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                    </h4>
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                </div>
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                <div id="P2-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P2">
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                    <div class="panel-body">
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<p>
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<b>Materials:</b><br>
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5 ml LB broth<br>
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5 μl antibiotic<br>
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Loops<br>
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12 ml culture tube<br>
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<br>
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<b>Methods:</b><br>
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Overnight cultures were prepared under sterile conditions using a Bunsen burner<br>
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<ol style="font-size:16px;">
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<li>Add 5 ml liquid LB media into 12 ml culture tubes</li>
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<li>Add 5 μl of appropriate antibiotic into the broth</li>
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<li>Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth</li>
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<li>Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm</li>
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</ol>
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</p>
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  </div>
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</div>
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</div>
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<div class="some-padding"></div>
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<div class="some-padding"></div>
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<h2>Paracoccus denitrificans protocols</h2>
 
<h2>Paracoccus denitrificans protocols</h2>

Revision as of 21:43, 31 October 2017



Protocols


On this page, the ecolibrium team would like to share with you the protocols that we have been using over the summer. This library of protocols has been developed alongside our supervisors for the purpose of this study. Here you can find the exact methods we use to generate our data and results. We share them in the interest of reproducibility.

Materials:
LB broth
Ice
Selection plates

Methods:

  1. Thaw 50µL competent E. coli cells on ice for 10 minutes
  2. Add:
    • 5-10 µl DNA from a ligation reaction mix or
    • 10-100ng DNA of a known plasmid
  3. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  4. Place the mixture on ice for 30 minutes. Do not mix.
  5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  6. Place on ice for 5 minutes. Do not mix.
  7. Pipette 950 µl of room temperature SOC or LB media into the mixture.
  8. Incubate at 37°C and 200-250 rpm for 60 minutes.
  9. Mix the cells thoroughly by flicking the tube and inverting.
  10. Spread:
    • For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
      1. Pellet cells at 8000rpm for 3 minutes
      2. Remove and dispense 600 µL of supernatant
      3. Re-suspend cells by light vortexing
      4. Plate resuspended cells as above
    • For known plasmid: 10 & 100 µL of each transformation reaction onto a selection plate. For the rest of 890 µL:
      1. Pellet cells at 8000rpm for 3 minutes
      2. Remove and dispense 600 µL of supernatant
      3. Re-suspend cells by light vortexing
      4. Plate resuspended cells as above
  11. Incubate overnight at 37°C with plates upside down.

Materials:
5 ml LB broth
5 μl antibiotic
Loops
12 ml culture tube

Methods:
Overnight cultures were prepared under sterile conditions using a Bunsen burner

  1. Add 5 ml liquid LB media into 12 ml culture tubes
  2. Add 5 μl of appropriate antibiotic into the broth
  3. Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth
  4. Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm

Paracoccus denitrificans protocols


1. Grow Paracoccus cultures in liquid media 2. Dilute 500mL of Paracoccus denitrificans culture in 500mL SGM17 medium ***COLD ROOM FROM HERE*** 3.Harvest by centrifuge 250mL samples each in 2 four centrifuge bottles at 5000xg at 4 C. Carefully dump out supernatant. 4.Completely resuspend (shake vigorously without up & down motion to avoid liquid loss) the pellets from the previous step in all centrifuge bottles each with 10050mL of 0.5 M sucrose solution with 10% glycerol. 5. Combine the liquid(containing resuspended pellets) into one centrifuge bottle, add 200mL water to the other empty centrifuge bottle for counterbalance. 6. Centrifuge the two bottles at 5000xg at 4 C. 7. Dump out supernatant and resuspend the pellets with 100mL of 0.5 M sucrose solution with 10% glycerol. Counter-balance bottle should have 100mL of water. 8. Centrifuge the two bottles at 5000xg at 4 C. 9. Dump out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol. 10. Aliquot the final 10mL of liquid into 0.25mL(250uL) with 40 microcentrifuge tubes. 11. Flash freeze with liquid nitrogen and store at -80 C.


  • 1. Thaw and mix 50 uL portions cells with 5 uL DNA
  • 2. Transfer to ice cold electroporation cuvette
  • 3. Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms
  • 4. The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad
  • 5. IMMEDIATELY following electroporation: carefully mix suspension with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes
  • 6. Dilutions were made in the SGM17 media
  • 7. Incubate cells at 30 C for 2 hours
  • 8. Take 100 uL portions and plate.

    • Measurement protocols


    Isolate Single colonies
  • 1. Select glycerol stock cultures from -80 C storage to begin
  • 2. Thaw on ice for 15 minutes and observe when the cell stock begins to melt
  • 3. Take a plastic p-loop or other spreading instrument and spread on a selective media agar plate containing the appropriate antibiotics
  • If the culture is P. denitrificans, culture on PD media or standard LB
  • If the culture is E. coli use LB media
  • If using untransformed bacteria, equivalent non-selective media should be used
  • 4. Incubate both P. denitrificans and E. coli overnight at 37 C
  • 5. Isolate single colonies after overnight incubation and resuspend in 60 mL volumes of liquid selective LB media with appropriate antibiotics

    Preculture for Device Testing

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