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| − | <html class="-webkit-"><head><meta http-equiv="Content-Type" content="text/html; charset=UTF-8">
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| − | <head>
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| − | <link href="https://fonts.googleapis.com/css?family=Ubuntu+Condensed" rel="stylesheet">
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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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| − | <div class="menu-bar">
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab">HOME</a>
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| − | <div class="menu-item">
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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/notebook">PROTOCOLS</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/collaborations">COLLABORATIONS</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/communityengagement">COMMUNITY ENGAGEMENT</a>
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/applieddesign">APPLIED DESIGN</a>
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| − | <div class="menu-item">
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/integratedpractices">INTEGRATED PRACTICES</a>
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| − | <div class="menu-item">
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/awards">AWARDS</a>
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| − | <div class="menu-item">
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| − | <a href="https://2017.igem.org/Team:ULaVerne_Collab/safety">SAFETY</a>
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| − | </div>
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| − | <div class="menu-bar-border-bottom">
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| − | </div>
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| − | </div>
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| − |
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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">GENERAL PROTOCOLS</h2>
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| − | <div id="bodyText">
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| − | </div>
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| − | </div>
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| − | </section>
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">Transformation Protocol (E. coli)</h2>
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| − | <div id="bodyText">
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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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| − | </div>
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| − | </div>
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| − | </section>
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">Miniprep</h2>
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| − | <div id="bodyText">
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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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| − | </div>
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| − | </div>
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| − | </section>
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| − |
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">Digestion</h2>
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| − | <div id="bodyText">
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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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| − |
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| − | </div>
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| − | </div>
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| − | </section>
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| − |
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| − |
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">Ligation</h2>
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| − | <div id="bodyText">
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| − | </div>
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| − | </div>
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| − | </section>
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">QIAEX II Gel Extraction Kit Protocol</h2>
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| − | <div id="bodyText">
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| − | </div>
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| − | </div>
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| − | </section>
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">QIAquick PCR Purification Kit</h2>
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| − | <div id="bodyText">
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| − | 1. Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. <BR>
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| − | 2. Apply sample to QIAquick purple column and centrifuge at 13,000rpm for 1 minute. Discard flow through and place column back in the same tube.<BR>
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| − | 3. Add 750uL of Buffer PE to column and centrifuge at 13,000rpm for 1 minute. Discard flow through and place column back in same tube.<BR>
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| − | 4. Centrifuge empty column at 13,000 for 1 minute to remove residual wash buffer.<BR>
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| − | 5. Place column in a clean 1.5mL microfuge tube.<BR>
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| − | 6. Add 50uL nuclease-free water to center of membrane. Let column stand for 1 minute and centrifuge for 1 minute.<BR>
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| − | 7. Label microfuge tube containing purified vector.<BR>
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| − | </div>
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| − | </div>
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| − | </section>
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">CHLAMYDOMONAS PROTOCOLS</h2>
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| − | <div id="bodyText">
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| − | </div>
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| − | </div>
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| − | </section>
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| − |
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">Transformation Protocol (E. coli)</h2>
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| − | <div id="bodyText">
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| − | <u>Culturing and Preserving Chlamydomonas Reinhardtii </u><BR>
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| − | Prior to beginning the culturing of the Chlamydomonas<BR>
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| − | Place cells upright away from direct sunlight in a tube rack or beaker. Keep them in a cool area at room temperature until they are ready to be cultured<BR>
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| − | (DO NOT leave them unattended for more than three days. If they are not going to be treated within the first 36 hours of arrival, transfer the cell culture to a weak nutrient solution like half-strength Alga-Gro® Medium or minimal growth medium).<BR>
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| − | Autoclave anything that will be touching the cell culture (Erlenmeyer flasks, pipette tips, etc.) for 15 minutes at 15 pounds of pressure <BR>
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| − | Set up the (2) 40 watt light bulbs about 45 to 60 cm above the culture <BR>
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| − | Cover the area in foil to retain heat <BR>
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| − | Beginning the culture <BR>
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| − | In a sterile hood, pipette 200mL of TAP medium into a 500mL Erlenmeyer flask<BR>
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| − | Pipette 10mL of the Chlamydomonas strain into a 500mL Erlenmeyer flask containing the 200mL fresh medium (If the medium is grown in a slant tube, in a sterile field, use an inoculating loop to pull out some of the Chlamydomonas and place it in the 200 mL of fresh medium)<BR>
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| − | Parafilm seal the Erlenmeyer flask to prevent contamination and place the flask on a stir plate on top of the petri dish lid<BR>
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| − | Check it every so often to make sure there is continuous movement (YOU DO NOT WANT ANY SHADOW AREAS) <BR>
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| − | Set a timer to alternate between light and dark every 12 hours <BR>
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| − | Using a spectrophotometer measure the growth of the cell culture everyday at the same times (Set the spectrophotometer at 750nm and use identical Kimax tubes)<BR>
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| − | Incubate the cell culture until the growth curve reaches 0.5 <BR>
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| − | Once the cell culture reaches 0.5 on the spectrophotometer and the culture’s cell density of approximately 0.9-2.0 x 107 ml-1 (3-5 days after transfer, late linear phase), aliquot the cells into 1.5mL Eppendorf tubes <BR>
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| − | Have liquid nitrogen ready in a foam container to snap freeze the aliquots<BR>
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| − | </div>
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| − | </div>
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| − | </section>
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| − |
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">Miniprep</h2>
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| − | <div id="bodyText">
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| − |
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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>
| |
| − | 2. Resuspend pelleted bacterial cells in 250uL buffer P1.<BR>
| |
| − | 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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| − |
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| − | </div>
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| − | </div>
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| − | </section>
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| − |
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">Digestion</h2>
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| − | <div id="bodyText">
| |
| − |
| |
| − | 1. Thaw all enzymes need and aliquot appropriate amounts. <BR>
| |
| − | 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>
| |
| − | 3. Add appropriate amount of DNA into the nuclease free water. <BR>
| |
| − | 4. Add 5uL of NEB Buffer to each tube. <BR>
| |
| − | 5. Add 1uL of the first enzyme to each tube. (BsrG1) <BR>
| |
| − | 6. Add 1uL of the second enzyme to each tube. (BamHI) <BR>
| |
| − | 7. Add 1uL of SAP to vector tube.<BR>
| |
| − | 8. Agitate each solution to mix well.<BR>
| |
| − | 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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| − |
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| − | </div>
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| − | </div>
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| − | </section>
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| − |
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| − |
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">Ligation</h2>
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| − | <div id="bodyText">
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| − |
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| − | </div>
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| − | </div>
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| − | </section>
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| − |
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| − |
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">QIAEX II Gel Extraction Kit Protocol</h2>
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| − | <div id="bodyText">
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| − |
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| − | </div>
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| − | </div>
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| − | </section>
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| − |
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| − | <section class="more grid">
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| − | <div class="more-content">
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| − | <h2 class="content-title">QIAquick PCR Purification Kit</h2>
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| − | <div id="bodyText">
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| − |
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| − |
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| − |
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| − | </div>
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| − | </div>
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| − | </section>
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| − |
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| − |
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| − | <div class="hidden"><div id="mainLogo"> <img src=""> </div> </div>
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| − |
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| − |
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| − | </div>
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| − | </main>
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| − | </body>
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| − | </html>
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