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| − | <title>MAIN</title>
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| − | <center><img src="https://dl.dropbox.com/s/zux5t3n8hiswgvz/mainlogo2.png"></center>
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| − | <!-- * * * M E N U * * * -->
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab">HOME</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/team">TEAM</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/attributions">ATTRIBUTIONS</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/project">PROJECT</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/parts">PARTS</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/model">MODEL</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/integratedpractices">INTEGRATED PRACTICES</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/safety">SAFETY</a>
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| − | <main id="content" class="main-area">
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">PROTOCOLS</h2>
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| − | </section>
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| − | <h2 class="content-title">Transformation Protocol (E. coli)</h2>
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| − | 1. Pre-chill microcentrifuge tube and thaw competent cells on ice. <BR>
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| − | 2. Add 50uL of competent cells into the chilled microfuge tube. <BR>
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| − | 3. Add 4uL of DNA into the microcentrifuge tube. <BR>
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| − | 4. Incubate on ice on 30 minutes. <BR>
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| − | 5. Heat shock at 42 degrees Celsius for 45 sec. <BR>
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| − | 6. Incubate cells on ice for 2 minutes. <BR>
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| − | 7. Add 200uL of SOC media into the microcentrifuge tube. <BR>
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| − | 8.Incubate and shake at 37 degree Celsius for 1-2 hours. <BR>
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| − | 9. Pipette 200uL of cells into LB plate with ampicillin resistance. <BR>
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| − | 10. Incubate plate overnight at 37 degrees Celsius. <BR>
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| − | </section>
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| − | <h2 class="content-title">Miniprep</h2>
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| − | 1. Transfer 1.5mL of overnight bacterial cultures from conicol tubes to microcentrifuge tubes. Centrifuge at 8000rpm for 3 minutes at room temperature. Discard supernatant and repeat until all of the overnight bacterial culture is suspended. <BR>
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| − | 2. Resuspend pelleted bacterial cells in 250uL buffer P1.<BR>
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| − | 3. Add 250uL buffer P2 and mix by inverting 4-6 times. <BR>
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| − | 4. Add 350uL buffer N3 and mix by inverting.<BR>
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| − | 5. Centrifuge for 10 minutes at 1300rpm. <BR>
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| − | 6. Pipette the supernatant from step 5 to the QIAprep spin column. Centrifuge for 1 minute and discard flow through.<BR>
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| − | 7. Wash the QIAprep spin column by adding 750uL buffer PE. Centrifuge for 1 minute and discard flow through. <BR>
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| − | 8. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. Add 50uL nuclease free water to the center of the QIAprep spin column. Let it stand for 1 minute. Centrifuge for 1 minute. <BR>
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| − | </section>
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| − | <h2 class="content-title">Digestion</h2>
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| − | 1. Thaw all enzymes need and aliquot appropriate amounts. <BR>
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| − | 2. Pipette the amount of nuclease free water needed to make 500ug of DNA. (500 ug/Concentration. Then take that amount and subtract it from 50uL to get the amount of water needed) <BR>
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| − | 3. Add appropriate amount of DNA into the nuclease free water. <BR>
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| − | 4. Add 5uL of NEB Buffer to each tube. <BR>
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| − | 5. Add 1uL of the first enzyme to each tube. (BsrG1) <BR>
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| − | 6. Add 1uL of the second enzyme to each tube. (BamHI) <BR>
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| − | 7. Add 1uL of SAP to vector tube.<BR>
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| − | 8. Agitate each solution to mix well.<BR>
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| − | 9. Incubate parts at 37 degrees Celsius for 15 minutes. Incubate vector at 37 degrees Celsius for 30 minutes.<BR>
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| − | 10. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.<BR>
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| − | </div>
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| − | </section>
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| − | <div class="hidden"> <div id="mainLogo"> <img src=""> </div> </div>
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| − | </body>
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