Team:UC Davis/Notebook/Protocols

From 2012.igem.org

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<li>LB Broth</li>
<li>LB Broth</li>
<li>Ethylene Glycol Agar Plates</li>
<li>Ethylene Glycol Agar Plates</li>
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<li>Serological pipets (10mL) for cell re-suspension</li>
 
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<ul>
 
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<li>PipetAide</li>
 
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</ul>
 
</ul>
</ul>
<p>Procedure</p>
<p>Procedure</p>
<ul>
<ul>
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<li>Prepare a 10mL liquid culture in LB in a 50mL Conical tube and grow it overnight until it reaches OD~0.2.</li>
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<li>1. 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 <i>Eppendorf Centrifuge 5810 R</i> for 10 minutes.</li>
<li>Chill the cells on ice and spin down the 10mL aliquots at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 10 minutes.</li>
<li>Wash twice with 10mL Buffer A.</li>
<li>Wash twice with 10mL Buffer A.</li>
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<li>Pipet up and down to re-suspend pellet</li>
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            <ul>
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<li>Pellet cells as in step 2</li>
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                <li>Pipet up and down to mix, pellet cells, decant supernatant.</li>
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<li>Decant supernatant in waste</li>
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            </ul>
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<li>Bleach waste </li>
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<li>Re-suspend the pellet in 5mL of Buffer A and transfer the medium to a Falcon tube.</li>
 +
<li>Add (35μL, 70μL, or 105μL) of EMS into each tube of re-suspended cells. </li>
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<li>Close tubes and parafilm the lid 2X.</li>
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<li>Vortex to mix and place in a secondary container (50mL conical tube).</li>
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<li>Transfer to mixing platform and agitate at ~30 rpm at 37°C.</li>
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<li>Withdraw samples at fixed time points.</li>
 +
<li>At each time point, take 1mL aliquots of the sample and place in a 15mL Falcon tube. Place in a secondary container (50mL Conical tube) and spin at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 5 minutes. </li>
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<li>Discard supernatant in waste container. </li>
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<li>Wash twice in 5mL of Buffer A and discard supernatant in waste container.</li>
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<li>Re-suspend in 5mL of Buffer A and titer for viable cells.</li>
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<li>Add 0.5mL of mutagenized culture into 10mL of LB broth in a 50mL conical tube.</li>
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<li>Grow cultures overnight at 37°C and plate for mutants on EG agar plates.</li>
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<li>Look for viable cells</li>
 +
</ul>
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<p>Safety: Health Hazards</p>
 +
<ul>
 +
<li>Acute toxicity (oral, dermal, inhalation), category 4 </li>
 +
<li>Skin irritation, category 2 </li>
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<li>Eye irritation, category 2 </li>
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<li>Skin sensitization, category 1 </li>
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<li>Specific Target Organ Toxicity – Single exposure, category 3</li>
 +
<li>LD50 – 470mg/kg</li>
</ul>
</ul>
 +
 +
<p>Recommended Safety & Handling</p>
 +
<ul><li>Always work in fume hood and wear lab coat, goggles, and gloves.</li></ul>
 +
     </div>
     </div>

Revision as of 07:12, 30 September 2012

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