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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/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">GENERAL PROTOCOLS</h2>
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| − | <div id="bodyText">
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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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| − | <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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| − | 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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| − | </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">Ligation</h2>
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| − | <div id="bodyText">
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| − | 1. Find the 3:1 (insert:vector) molar ratio of DNA ends. Use NEB ligation calculator. <BR>
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| − | 2. Calculate amount of vector to 50ng vector. <BR>
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| − | 3. Add appropriate amount of nuclease free water into a microcentrifuge tube for a total of volume of 20uL. <BR>
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| − | 4. Add appropriate amount of each parts into the tube with water for a total volume of 20uL. <BR>
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| − | 5. Add 2 uL of 10X T4 DNA Ligase Buffer into the tube. <BR>
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| − | 6. Add 1 uL of T4 DNA Ligase into the tube. <BR>
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| − | 7. Incubate at room temperature for 10 minutes. <BR>
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| − | 8. Heat inactivate at 65 degrees Celsius for 10 minutes. <BR>
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| − | 9. Store in at -20 degrees Celsius freezer or transform ligated DNA. <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">QIAEX II Gel Extraction Kit Protocol</h2>
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| − | <div id="bodyText">
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| − | 1. Cut DNA band from the agarose gel with a clean, sharp razor blade. Weigh a 1.7mL centrifuge tube. <BR>
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| − | 2. Add the DNA gel slice to weighed tube. Reweigh and subtract from original tube to find weight of DNA slice.<BR>
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| − | 3. Add Buffer QX1 according to DNA fragment size: 6 volumes for <100bp; 3 volumes for 100bp-4kb; 3 volumes with 2 volumes of water for >4kb; 6 volumes when using >2% agarose gels.<BR>
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| − | 4. Resuspend QIAEX II by vortexing for 30s. Add QIAX II to the tube with the sample and vortex: Use 10µL of QIAEX II for ≤ 2µg DNA; 30µL for 2-10µg DNA; and an additional 30µL for each additional 10µg DNA.<BR>
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| − | 5. Incubate at 50°C for 10min vortexing every 2min to melt agarose and bind DNA. Keep an eye on color change. If so adjust accordingly.<BR>
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| − | 6. Centrifuge the sample for 30s and carefully remove supernatant.<BR>
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| − | 7. Wash the pellet with 500µL Buffer QX1. Resuspend by vortexing. Centrifuge for 30s and remove supernatant.<BR>
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| − | 8. Wash the pellet twice with 500µL Buffer PE. Resuspend pellet by vortexing. Centrifuge for 30s and remove supernatant.<BR>
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| − | 9. Air-dry the pellet until it becomes white. Do not vacuum dry.<BR>
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| − | 10. To elute DNA, add 20µL nuclease free water and resuspend the pellet by vortexing.* Incubate according to fragment size: 5min at room temp. for ≤4kb; 5min at 50°C for 4-10kb; 10min at 50°C for > 10kb.<BR>
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| − | *Fragments larger than 10kb resuspend by flicking and inverting tube as vortexing can cause shearing.<BR>
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| − | 11. Centrifuge for 30s and pipet supernatant into clean tube. This is your purified DNA.<BR>
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| − | 12. Repeat steps 9 and 10 to combine eluates and increase yield.<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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| − | <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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| − | <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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| − | 1. 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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| − | 2. 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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| − | 3. Set up the (2) 40 watt light bulbs about 45 to 60 cm above the culture <BR>
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| − | 4. Cover the area in foil to retain heat <BR><BR>
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| − | <u>Beginning the culture </u> <BR>
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| − | 5. In a sterile hood, pipette 200mL of TAP medium into a 500mL Erlenmeyer flask<BR>
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| − | 6. 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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| − | 7. 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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| − | 8. Check it every so often to make sure there is continuous movement (YOU DO NOT WANT ANY SHADOW AREAS) <BR>
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| − | 9. Set a timer to alternate between light and dark every 12 hours <BR>
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| − | 10. 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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| − | 11. Incubate the cell culture until the growth curve reaches 0.5 <BR>
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| − | 12. 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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| − | <i>Have liquid nitrogen ready in a foam container to snap freeze the aliquots</i><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">Culturing Chlamydomonas on solid medium
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| − | </h2>
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| − | <div id="bodyText">
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| − | 1. Set up a sterile field <BR>
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| − | 2. Add 100uL of the liquid culture to TAP agar plates using a sterile rod/glass beads<BR>
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| − | 3. Allow the plates to dry in the sterile field <BR>
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| − | 4. Seal the plates in parafilm (this helps reduce the contamination)<BR>
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| − | 5. Place the plates in the room temperature (25 degrees Celsius) incubator overnight <BR>
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| − | 6. Remove the plates from the incubator and place them in a 4 degrees celsius refrigerator to prevent further growth<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">Glass Bead Transformation of Chlamydomonas</h2>
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| − | <div id="bodyText">
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| − |
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| − | <u>Preparation</u> <BR>
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| − | 1. Grow cell culture to an OD750 of 0.15 to 0.4<BR>
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| − | 2. Centrifuge at 400g for 5mins at room temperature
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| − | 3. Resuspend in 1/100th the original volume of TAP
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| − | 4. Add the following into a separate tube:<BR>
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| − |     a. 8000PEG to 5% final concentration (10mg at 1mL of water is 1%)<BR>
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| − |     b. 3ug of DNA<BR>
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| − |     c. 0.3g of 500micron glass beads<BR>
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| − |     d. 0.4mL Chlamy cell suspension<BR>
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| − | 5. Mix with a pipette<BR>
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| − | 6. Vortex at max speed for 15 seconds<BR><BR>
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| − | <u>Plating and Selection</u><BR>
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| − | 1. Take 25uL of the cell suspension and add to 100uL of TAP with an appropriate antibiotic<BR>
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| − | 2. Spread on a TAP plate with an appropriate antibiotic, using large glass beads<BR>
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| − | 3. Allow the liquid to dry while avoiding light<BR>
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| − | 4. Seal the plates with parafilm<BR>
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| − | 5. Allow the colonies to grow (1-3 weeks)<BR>
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| − | 6. Transfer the remaining cell/vortex culture to a 125mL flask with 20mL of TAP (w/o antibiotic)<BR>
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| − | 7. Incubate for 6 hours on an orbital shaker at 70rpm<BR>
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| − | 8. Add antibiotic to an appropriate concentration<BR>
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| − | 9. Take 50uL of the cell suspension and spread on a TAP plate with an appropriate antibiotic with large glass beads<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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| − | <h2 class="content-title">OD Spectrophotometer Readings (Prepare cuvettes in a Sterile Hood)</h2>
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| − | <div id="bodyText">
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| − |
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| − | 1. Set wavelength on a biochrom spectrophotometer to 750 nm <BR>
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| − | 2. Blank spectrophotometer before each reading<BR>
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| − |     a. Inside of a sterile hood pipette 3mL of TAP Media into 1 cuvette<BR>
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| − |     b. Take cuvette with medium outside of hood<BR>
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| − |     c. Wipe outside of cuvette thoroughly with KIMwipe<BR>
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| − |     d. Insert into spectrophotometer and hit blank<BR>
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| − | 3. OD Readings
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| − |     a. Inside of a sterile hood pipette 2mL of TAP Media and 1mL of Chlamydomonas into one cuvette (3x)<BR>
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| − |     b. Outside of the hood wipe the outside of the cuvette thoroughly with a KIMwipe and insert into spectrophotometer<BR>
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| − |     c. Directly prior to reading, resuspend cells with pipette <BR>
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| − |     d. Take reading<BR>
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| − |     e. Multiply reading by three<BR>
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| − |     f. Calculate the average <BR>
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| − | </div>
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