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| - | <h1>Transforming Competent Cells</h1><BR><BR>
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| - | <ol style="list-style-type:decimal;">
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| - | <li>Thaw the DH5-alpha cells (from -80ºC freezer), plasmid sample (from -20ºC freezer), and the antibiotic stock (from -20ºC freezer) ON ICE.<BR><BR>
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| - | <li>Split the thawed cells into 2x 100µl aliquots. <BR><BR>
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| - | <li>To each aliquot add 1µl of plasmid DNA.<BR><BR>
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| - | <li>Incubate ON ICE for ~30 minutes. Incubation can be longer than this but certainly NO shorter. <BR><BR>
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| - | <li>During this incubation time prepare the antibiotic agar plates: <BR><BR>
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| - | <li>Melt the agar jar for 15 minutes with the microwave on ‘defrost’. Check on the bottle every 5 or so minutes to ensure it is not overflowing.<BR><BR>
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| - | <li>Cool the agar bottle in 67ºC water bath. During this time switch on the the laminar flow hood to create a sterile environment.<BR><BR>
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| - | <li>Once cool take the agar and petri dishes to the laminar flow hood.<BR><BR>
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| - | <li>Add the appropriate volume of antibiotic to the agar bottle before pouring.<BR><BR>
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| - | <li>Pour the agar evenly to no higher than the raised line. NOTE: place the lid resting on the edge of the dish so as to catch any drops that fall from the bottle.<BR><BR>
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| - | <li>Leave the agar plates to set (~15-30 minutes)<BR><BR>
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| - | <li>Heat shock the bacteria by placing them in the 42ºC water bath for NO MORE THAN 45 SECONDS. <BR><BR>
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| - | <li>Immediately place the bacteria on ICE for 1 minute. <BR><BR>
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| - | <li>Add 800µl LB and incubate at 37ºC for 1 hour. <BR><BR>
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| - | <li>Plate the bacteria under sterile conditions (bunsen burner ON): <BR><BR>
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| - | <li>Place a glass spreader in ethanol. <BR><BR>
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| - | <li>Flame the spreader to burn off the ethanol and let it cool. <BR><BR>
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| - | <li>Plate #1: spread 100µl of cell culture onto the plate<BR><BR>
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| - | <li>Plate #2: spin down the remaining cells from step 3. Discard the supernatant and resuspend the pellet in 100µl of fresh culture. Spread this onto a second plate as in previous step<BR><BR>
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| - | </ol>
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| - | <li>Incubate at 37ºC overnight. </li><BR><BR>
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