Team:UC Davis/Notebook/Protocols

From 2012.igem.org

(Difference between revisions)
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         </ul>
         </ul>
         </li>
         </li>
-
         <li class="selected"><a href="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
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         <li class="selected"><a title="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
           <ul>
           <ul>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook_Overview ">Notebook Overview</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook_Overview ">Notebook Overview</a></li>
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         <li ><a href="https://2012.igem.org/Team:UC_Davis/Safety" title="Safety">Safety</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Safety" title="Safety">Safety</a></li>
-
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
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         <li ><a title="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
           <ul>
           <ul>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a></li>
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<p></p>
<p></p>
</div>
</div>
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<div id="myleftrightbox"  class="fourboxes-3">
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<div id="myleftrightbox"  class="fourboxes-2">
<a href="https://2012.igem.org/Team:UC_Davis/Project/Notebook/Gallery"><img src="http://img.photobucket.com/albums/v26/bluemelon/gallery_small_banner.jpg"></a>
<a href="https://2012.igem.org/Team:UC_Davis/Project/Notebook/Gallery"><img src="http://img.photobucket.com/albums/v26/bluemelon/gallery_small_banner.jpg"></a>
</div>
</div>
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   <p class="menu_head"> Registry Part Distribution Rehydration </p>
   <p class="menu_head"> Registry Part Distribution Rehydration </p>
     <div class="menu_body">
     <div class="menu_body">
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•Add 20 µL sterile H2O to desired well in distribution plate.
+
<ul>
-
<br>•Incubate at room temperature for ~10 minutes.
+
<li>Add 20 µL sterile H2O to desired well in distribution plate.</li>
-
<br>•Transfer resuspension to microcentrifuge tube.
+
<li>Incubate at room temperature for ~10 minutes.</li>
 +
<li>Transfer resuspension to microcentrifuge tube.</li>
 +
</ul>
     </div>
     </div>
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     <div class="menu_body">
     <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
-
•Competent Cells
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<ul>
-
<br>•DNA template
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<li>Competent Cells</li>
-
<br>•800 µL LB
+
<li>DNA template</li>
-
<br>•LB+Antibiotic Plates
+
<li>800 µL LB</li>
 +
<li>LB+Antibiotic Plates</li>
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Thaw competent cells on ice.
+
<ul>
-
<br>•Transfer 50 µL of competent cells to chilled falcon tubes.
+
<li>Thaw competent cells on ice.</li>
-
<br>•Add 1 µL of template to cells (2.5 µL if dilute).
+
<li>Transfer 50 µL of competent cells to chilled falcon tubes.</li>
-
<br>•Incubate on ice for 30 minutes.
+
<li>Add 1 µL of template to cells (2.5 µL if dilute).</li>
-
<br>•Heat schock in 42 °C water bath for 90 seconds.
+
<li>Incubate on ice for 30 minutes.</li>
-
<br>•Immediately place back onto ice for 2 minutes.
+
<li>Heat schock in 42 °C water bath for 90 seconds.</li>
-
<br>•Add 800 µL of LB to each tube.
+
<li>Immediately place back onto ice for 2 minutes.</li>
-
<br>•Incubate at 37 °C for 1 hour.
+
<li>Add 800 µL of LB to each tube.</li>
-
<br>•Place 200 µL of the transformed cells on plates containing LB and the appropriate antibiotic.
+
<li>Incubate at 37 °C for 1 hour.</li>
-
<br>•Incubate overnight at 37 °C.
+
<li>Place 200 µL of the transformed cells on plates containing LB and the appropriate antibiotic.</li>
 +
<li>Incubate overnight at 37 °C.</li>
 +
</ul>
     </div>
     </div>
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     <div class="menu_body">
     <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
-
•22 µL dH20
+
<ul>
-
<br>•1 µL BSA
+
<li>22 µL dH20
-
<br>•5 µLBuffer
+
<li>1 µL BSA
-
<br>•20 µL Template
+
<li>5 µLBuffer
-
<br>•1 µL Enzyme 1
+
<li>20 µL Template
-
<br>•1 µL Enzyme 2
+
<li>1 µL Enzyme 1
 +
<li>1 µL Enzyme 2
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Mix reactants, adding enzyme last.
+
<ul>
-
<br>•Place at 37 C for 3-5 hours.
+
<li>Mix reactants, adding enzyme last.
-
<br>•If not purifying or running on a gel immediately, increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme).
+
<li>Place at 37 C for 3-5 hours.
-
<br>•Run on a gel and extract product.
+
<li>If not purifying or running on a gel immediately, increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme).
 +
<li>Run on a gel and extract product.
 +
</ul>
     </div>
     </div>
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     <div class="menu_body">
     <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
-
•10 uL Q Solution
+
<ul>
-
<br>•5 µL 10x Buffer
+
<li>10 uL Q Solution
-
<br>•1.25 µL 10mM dNTPs
+
<li>5 µL 10x Buffer
-
<br>•1 µL Template
+
<li>1.25 µL 10mM dNTPs
-
<br>•1 µL Forward Primer
+
<li>1 µL Template
-
<br>•1 µL Reverse Primer
+
<li>1 µL Forward Primer
-
<br>•0.3 µL Taq
+
<li>1 µL Reverse Primer
-
<br>•0.15 µL PFU
+
<li>0.3 µL Taq
-
<br>•30 µL dH20
+
<li>0.15 µL PFU
 +
<li>30 µL dH20
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.
+
<ul>
-
<br>•Run in thermal cycler.
+
<li>Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.
 +
<li>Run in thermal cycler.
 +
</ul>
   </div>
   </div>
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     <div class="menu_body">
     <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
-
•10 µL Q Solution
+
<ul>
-
<br>•5 µL 10x Buffer
+
<li>10 µL Q Solution
-
<br>•1.25 µL 10mM dNTPs
+
<li>5 µL 10x Buffer
-
<br>•2.5 µL Forward Primer
+
<li>1.25 µL 10mM dNTPs
-
<br>•2.5 µL Reverse Primer
+
<li>2.5 µL Forward Primer
-
<br>•0.3 µL Taq
+
<li>2.5 µL Reverse Primer
-
<br>•0.15 µL PFU
+
<li>0.3 µL Taq
-
<br>Template based on concentrations determined by SOEing calculator: “Link”
+
<li>0.15 µL PFU
-
<br>±18.30 µL dH20 based on concentrations of template (above) by SOEing calculator: “Link again”
+
<li>Template based on concentrations determined by SOEing calculator: “Link”
 +
<li>±18.30 µL dH20 based on concentrations of template (above) by SOEing calculator: “Link again”
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Mix reactants into PCR tubes.  
+
<ul>
-
<br>•Run in thermal cycler.
+
<li>Mix reactants into PCR tubes.  
-
<br>•Continue PCR SOEing of parts until completed.
+
<li>Run in thermal cycler.
 +
<li>Continue PCR SOEing of parts until completed.
 +
</ul>
     </div>
     </div>
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     <div class="menu_body">
     <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
-
•Digested Vector
+
<ul>
-
<br>•Digested Insert
+
<li>Digested Vector
-
<br>•Water
+
<li>Digested Insert
-
<br>•T4 DNA Ligase
+
<li>Water
-
<br>•T4 DNA Ligase Buffer
+
<li>T4 DNA Ligase
 +
<li>T4 DNA Ligase Buffer
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Mix these materials in the amounts determined by the reaction volume calculator <a href="https://static.igem.org/mediawiki/2011/7/73/UC_Davis_Reaction_Volume_Calculator.xls">here</a>.
+
<ul>
 +
<li>Mix these materials in the amounts determined by the reaction volume calculator <a href="https://static.igem.org/mediawiki/2011/7/73/UC_Davis_Reaction_Volume_Calculator.xls">here</a>.
 +
</ul>
     </div>
     </div>
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     <div class="menu_body">
     <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
-
•34 mM NaH<sub>2</sub>PO<sub>4</sub>
+
<ul>
-
<br>•64 mM K<sub>2</sub>HPO<sub>4</sub>
+
<li>34 mM NaH<sub>2</sub>PO<sub>4</sub>
-
<br>•20 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
+
<li>64 mM K<sub>2</sub>HPO<sub>4</sub>
-
<br>•1 µM FeSO<sub>4</sub>
+
<li>20 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
-
<br>•0.1 mM MgSO<sub>4</sub>
+
<li>1 µM FeSO<sub>4</sub>
-
<br>•10 µM CaCl<sub>2</sub>
+
<li>0.1 mM MgSO<sub>4</sub>
-
<br>•30 mM Ethylene Glycol
+
<li>10 µM CaCl<sub>2</sub>
-
<br>•30 mM Glycolate
+
<li>30 mM Ethylene Glycol
-
<br>•0.5% wt/vol Casein Acid Hydrolysate
+
<li>30 mM Glycolate
-
<br>•1.5% wt/vol Agar (if making solid media)
+
<li>0.5% wt/vol Casein Acid Hydrolysate
 +
<li>1.5% wt/vol Agar (if making solid media)
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.  
+
<ul>
-
<br>•Mix together NaH<sub>2</sub>PO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub>, (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, FeSO<sub>4</sub>, MgSO<sub>4</sub>, and CaCl<sub>2</sub>.  
+
<li>Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.  
-
<br>•Titrate to pH 7 with HCl.  
+
<li>Mix together NaH<sub>2</sub>PO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub>, (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, FeSO<sub>4</sub>, MgSO<sub>4</sub>, and CaCl<sub>2</sub>.  
-
<br>•Add the glycolate and casein acid hydrolysate.  
+
<li>Titrate to pH 7 with HCl.  
-
<br>•Mix in the ethylene glycol in a fume hood.  
+
<li>Add the glycolate and casein acid hydrolysate.  
-
<br>•If making solid media, also mix in agar.  
+
<li>Mix in the ethylene glycol in a fume hood.  
-
<br>•Autoclave on an appropriate cycle.     
+
<li>If making solid media, also mix in agar.  
 +
<li>Autoclave on an appropriate cycle.     
 +
</ul>
     </div>
     </div>
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     <div class="menu_body">
     <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
-
Luria Broth Media
+
<ul>
-
<br>Ethylene Glycol
+
<li>Luria Broth Media
-
<br>Tecan M200 Pro
+
<li>Ethylene Glycol
-
<br>E. <I>coli</I>
+
<li>Tecan M200 Pro
 +
<li>E. <I>coli</I>
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.  
+
<ul>
-
<br>1. Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
+
<li>Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.  
-
<br>2. Incubate at 37°C overnight on a shaker at 150 rpm.
+
<li>Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
-
<br>3. From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.  
+
<li>Incubate at 37°C overnight on a shaker at 150 rpm.
-
<br>4. Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
+
<li>From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.  
-
<br>5. Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.  
+
<li>Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
-
<br>6. Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
+
<li>Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.  
-
<br>7. With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.  
+
<li>Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
-
<br>8. Add “blank” wells of just LB media to an entire column to serve as a control for the LB.  
+
<li>With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.  
-
<br>9. Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.  
+
<li>Add “blank” wells of just LB media to an entire column to serve as a control for the LB.  
-
<br>10. Aliquot the remaining dilutions as the diagram below depicts.  
+
<li>Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.  
-
<br>11. Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.  
+
<li>Aliquot the remaining dilutions as the diagram below depicts.  
-
<br>12. Follow the steps of your Tecan machine appropriate to the specific bacterial strain.  
+
<li>Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.  
 +
<li>Follow the steps of your Tecan machine appropriate to the specific bacterial strain.  
 +
</ul>
     </div>  
     </div>  
 +
 +
  <p class="menu_head"> EMS (Ethyl Methane Sulfonate Mutagenesis)</p>
 +
    <div class="menu_body">
 +
<p>Materials</p>
 +
<ul>
 +
<li>Buffer A: Minimal medium + 0.2 M Tris (pH 7.5). per liter:
 +
<ul>
 +
<li>K<sub>2</sub>HPO<sub>4</sub>: 10.5g</li>
 +
<li>KH<sub>2</sub>PO<sub>4</sub>: 4.5g</li>
 +
<li>(NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>: 1g</li>
 +
<li>Sodium Citrate * 2H<sub>2</sub>O: 0.5g</li>
 +
<li>1M Tris (pH 7.5): 200mL Tris</li>
 +
</ul>
 +
</li>
 +
<li>EMS (Sigma)</li>
 +
<li>50mL Conical Corning tubes</li>
 +
<li>15mL Falcon tubes</li>
 +
<li>LB Broth</li>
 +
<li>Ethylene Glycol Agar Plates</li>
 +
<li>Serological pipets (10mL) for cell re-suspension</li>
 +
<ul>
 +
<li>PipetAide</li>
 +
</ul>
 +
</ul>
 +
<p>Procedure</p>
 +
<ul>
 +
<li>Prepare a 10mL liquid culture in LB in a 50mL Conical tube and grow it overnight until it reaches OD~0.2.</li>
 +
<li>Chill the cells on ice and spin down the 10mL aliquots at max speed in Eppendorf Centrifuge 5810 R for 10 minutes.</li>
 +
<li>Wash twice with 10mL Buffer A.</li>
 +
<li>Pipet up and down to re-suspend pellet</li>
 +
<li>Pellet cells as in step 2</li>
 +
<li>Decant supernatant in waste</li>
 +
<li>Bleach waste </li>
 +
 +
</ul>
 +
    </div>
 +
</div>
</div>
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