Team:UC Davis/Notebook/Protocols
From 2012.igem.org
(Difference between revisions)
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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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</ul> | </ul> | ||
<p>Procedure</p> | <p>Procedure</p> | ||
<ul> | <ul> | ||
- | <li>Prepare a 10mL liquid culture in LB in a 50mL Conical tube and grow it overnight until it reaches OD | + | <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> | ||
- | + | <ul> | |
- | <li> | + | <li>Pipet up and down to mix, pellet cells, decant supernatant.</li> |
- | <li> | + | </ul> |
- | <li> | + | <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> | ||
+ | <li>Close tubes and parafilm the lid 2X.</li> | ||
+ | <li>Vortex to mix and place in a secondary container (50mL conical tube).</li> | ||
+ | <li>Transfer to mixing platform and agitate at ~30 rpm at 37°C.</li> | ||
+ | <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> | ||
+ | <li>Discard supernatant in waste container. </li> | ||
+ | <li>Wash twice in 5mL of Buffer A and discard supernatant in waste container.</li> | ||
+ | <li>Re-suspend in 5mL of Buffer A and titer for viable cells.</li> | ||
+ | <li>Add 0.5mL of mutagenized culture into 10mL of LB broth in a 50mL conical tube.</li> | ||
+ | <li>Grow cultures overnight at 37°C and plate for mutants on EG agar plates.</li> | ||
+ | <li>Look for viable cells</li> | ||
+ | </ul> | ||
+ | <p>Safety: Health Hazards</p> | ||
+ | <ul> | ||
+ | <li>Acute toxicity (oral, dermal, inhalation), category 4 </li> | ||
+ | <li>Skin irritation, category 2 </li> | ||
+ | <li>Eye irritation, category 2 </li> | ||
+ | <li>Skin sensitization, category 1 </li> | ||
+ | <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