|
|
| (41 intermediate revisions by 4 users not shown) |
| Line 1: |
Line 1: |
| − | {{Team:Calgary/BasicPage
| + | MOVED |
| − | | + | |
| − | |INFOBOX=
| + | |
| − | <html>
| + | |
| − | | + | |
| − | </html>
| + | |
| − | | + | |
| − | |CONTENT=
| + | |
| − | <html>
| + | |
| − | | + | |
| − | <h1>Our Experiments</h1>
| + | |
| − | | + | |
| − | <h2>General Protocols</h2>
| + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Rehydration of Registry DNA <i class="fa fa-chevron-down"></i> </b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Registry DNA was rehydrated for completion of the Interlab Study. Also, Part BBa_K934001 (phaC1-A-B1) was rehydrated by the Secretion subgroup and transformed into our chassis so that P(3HB) was produced and preliminary secretion assays could be performed before the Synthesis subgroup had completed their cloning.</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>iGEM 2017 distribution kit</p>
| + | |
| − | <p>ddH₂O</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>Add 10 μL of ddH₂O to the desired well.</li>
| + | |
| − | <li>Pipette up and down 3-5 times.</li>
| + | |
| − | <li>Incubate at room temperature for 10 minutes.</li>
| + | |
| − | <li>Transform cells with 1 μL of rehydrated DNA. Store the remaining amount at -20°C.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Rehydration of IDT Synthesized DNA <i class="fa fa-chevron-down"></i></b> </h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Our genetic parts were ordered from IDT and arrived as a dry, lyophilized powder. They were resuspended in aqueous solution for cloning into pSB1c3 or pET29B vectors and to ligate multiple parts together. </p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Synthesized DNA from IDT (gBlocks)</p>
| + | |
| − | <p>ddH₂O</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <a name="plasmid_miniprep"><p><b>Protocol</b></p></a>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>Centrifuge tube containing the synthesized DNA for 3-5 seconds at 3000g to ensure that all material is at the bottom of the tube.</li>
| + | |
| − | <li>Add ddH₂O to reach a final concentration of 50 ng/μL.</li>
| + | |
| − | <li>Vortex.</li>
| + | |
| − | <li>Incubate at 50°C for 20 minutes.</li>
| + | |
| − | <li>Briefly vortex and centrifuge . Store at -80°C.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Preparation of Agar with Antibiotics <i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Antibiotics were added to agar to select for successful <i>E.coli</i> transformants. The vector pSB1c3 was selected for with chloramphenicol and pET29B was selected for with kanamycin. </p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Luria-Bertani broth with agar:</p>
| + | |
| − | <ul>
| + | |
| − | <li style="margin-left: 6%;"><p>10% (w/v) tryptone</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>5% (w/v) NaCl</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>10% (w/v) yeast extract</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>15% (w/v) agar</p></li>
| + | |
| − | </ul>
| + | |
| − | <p>Appropriate antibiotic:</p>
| + | |
| − | <ul>
| + | |
| − | <li style="margin-left: 6%;"><p>kanamycin (final concentration of 100 μg/mL)</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>chloramphenicol (final concentration of 30 μg/mL)</p></li>
| + | |
| − | </ul>
| + | |
| − | <p>dH2O</p>
| + | |
| − | <p>1500-mL Erlenmeyer flask</p>
| + | |
| − | <p>Stir bar</p>
| + | |
| − | <p>Aluminum foil</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>In a 1500-mL Erlenmeyer flask add 10g tryptone, 5 g yeast extract, 10g NaCl and 15g agar. Dissolve solids in 1000 mL dH2O and add a stir bar.</li>
| + | |
| − | <li>Cover flask loosely with aluminum foil, secure with autoclave tape and sterilize by autoclaving for 20 minutes. </li>
| + | |
| − | <li>Remove agar from autoclave using oven mitts. Allow agar to cool until warm to the touch before adding appropriate antibiotic.</li>
| + | |
| − | <li>Stir on hot plate and magnetic stirrer for 30 seconds.</li>
| + | |
| − | <li>Pour agar into plates using aseptic technique. </li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Plating Culture Broth on Agar Plates <i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Culture broth was plated on agar to isolate single colonies of <i>E.coli.</i></p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Luria-Bertani agar plate with appropriate antibiotic (if required)</p>
| + | |
| − | <p>Overnight culture of desired bacteria</p>
| + | |
| − | <p>70% ethanol</p>
| + | |
| − | <p>Spreading rod</p>
| + | |
| − | <p>Bunsen burner</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>Using aseptic technique pipette 50-100 μL of bacterial culture onto agar plate.</li>
| + | |
| − | <li>Dip spreading rod in 70% ethanol, pass over flame, and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture.</li>
| + | |
| − | <li>Use rod to spread bacterial culture over entire plate, spinning the plate at the same time.</li>
| + | |
| − | <li>Dip spreading rod in 70% ethanol, pass over flame, and allow excess liquid to burn off.</li>
| + | |
| − | <li>Incubate plates at 37°C overnight or until growth is observed.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Streaking Agar Plates <i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Culture broth was streaked on agar to isolate single colonies of <i>E.coli.</i></p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Luria-Bertani agar plate with appropriate antibiotic (if required)</p>
| + | |
| − | <p>Overnight culture of desired bacteria or single isolated colony on agar plate</p>
| + | |
| − | <p>Inoculation loop</p>
| + | |
| − | <p>Bunsen burner</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>Using aseptic technique, flame inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.</li>
| + | |
| − | <li>Dip the inoculation loop in bacterial culture or touch a single colony and streak the loop on ¼ of the surface of agar in a zigzag motion.</li>
| + | |
| − | <li>Flame the inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.</li>
| + | |
| − | <li>Run the cooled inoculation loop through one of the previous streaks ONCE, then streak 1/4 of the surface of the agar.</li>
| + | |
| − | <li>Repeat Steps 3 and 4 two more times.</li>
| + | |
| − | <li>Flame the inoculation loop until red hot. Allow it to cool for 10 seconds.</li>
| + | |
| − | <li>Incubate plates at 37°C overnight or until growth is observed.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Plasmid Miniprep from <i>Escherichia coli</i> <i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p><i>E. coli</i> DH5ɑ and BL21 were lysed and the pSB1c3 or pET29B vectors were isolated to be used in cloning our genetic constructs. Bacterial clones were lysed for analysis (eg: confirmation <a href="https://2017.igem.org/Team:Calgary/Experiments/imposter#restriction_digest">restriction digest</a>, genetic sequencing).</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>2.5 mL overnight culture of bacteria in Luria-Bertani broth with appropriate buffer in 16x125mm culture tube</p>
| + | |
| − | <p>Resuspension buffer (stored at 4°C):</p>
| + | |
| − | <ul>
| + | |
| − | <li style="margin-left: 6%;"> <p>50 mM Tris-HCl, pH 8</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>10 mM EDTA</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>100 μg/mL RNase A</p></li>
| + | |
| − | </ul>
| + | |
| − | <p>Lysis buffer:</p>
| + | |
| − | <ul>
| + | |
| − | <li style="margin-left: 6%;"><p>200 mM NaOH</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>1% (v/v) SDS</p></li>
| + | |
| − | </ul>
| + | |
| − | <p>Precipitation buffer:</p>
| + | |
| − | <ul>
| + | |
| − | <li style="margin-left: 6%;"><p>3 M CH₃CO₂K, pH 5.5</p></li>
| + | |
| − | </ul>
| + | |
| − | <p>Isopropanol</p>
| + | |
| − | <p>70% ethanol</p>
| + | |
| − | <p>Table-top centrifuge</p>
| + | |
| − | <p>Ice bucket</p>
| + | |
| − | <p>2-mL microcentrifuge tubes</p>
| + | |
| − | <p>1.5-mL microcentrifuge tubes</p>
| + | |
| − | <p>ddH₂O 3</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>Transfer 2 mL of the overnight culture to a 2-mL microcentrifuge tube and pellet the cells by spinning at 3500 g for 1 minute. Discard supernatant.</li>
| + | |
| − | <li>Resuspend pellet in 300 μL Resuspension buffer.</li>
| + | |
| − | <li>Add 300 μL Lysis buffer. Invert gently and incubate at room temperature for 3-5 minutes.</li>
| + | |
| − | <li>Add 300 μL Precipitation buffer. Invert gently. A white precipitate should form.</li>
| + | |
| − | <li>Centrifuge at 14,000g for 10 minutes at room temperature.</li>
| + | |
| − | <li>Retain supernatant in a clean 1.5-mL microcentrifuge tube.</li>
| + | |
| − | <li>Add 650 μL isopropanol. Gently invert and incubate at room temperature for 10 minutes.</li>
| + | |
| − | <li>Centrifuge at 14,000g for 10 minutes at 4°C. Discard supernatant.</li>
| + | |
| − | <li>Wash pellet with 500 μL cold 70% ethanol. Add to microcentrifuge tube. Do not resuspend.</li>
| + | |
| − | <li>Centrifuge at 14,000g for 5 minutes at 4°C. Discard supernatant.</li>
| + | |
| − | <li>Dry pellet in speed vac for 15-30 minutes, or until no more liquid remains visible in the tube.</li>
| + | |
| − | <li>Resuspend pellet in ddH₂O and store at - 20°C.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Restriction Digest <i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Our genetic parts and vectors were digested with restriction enzymes before they were ligated. Plasmids isolated from transformants (through <a href=" https://2017.igem.org/Team:Calgary/Experiments/imposter#plasmid_miniprep">Plasmid Miniprep</a>) were also digested for confirmation of ligation and transformation. </p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>DNA (eg: from plasmid miniprep)</p>
| + | |
| − | <p>Restriction enzymes</p>
| + | |
| − | <p>10X appropriate buffer</p>
| + | |
| − | <p>ddH₂O</p>
| + | |
| − | <p>100X Bovine Serum Albumin (BSA) (if using PstI)</p>
| + | |
| − | <p>0.2-mL PCR tubes</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>Into a 0.2-mL PCR tube add the following:</li>
| + | |
| − | <ul>
| + | |
| − | <li style="margin-left: 6%;"> <p>1 μg DNA</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>1 μL restriction enzyme 1</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>1 μL restriction enzyme 2</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>2 μL 10X appropriate buffer</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>1 μL 100X BSA, if using</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>ddH₂O to a final volume of 20 μL</p></li>
| + | |
| − | </ul>
| + | |
| − | <li>Incubate at 37°C for one hour.</li>
| + | |
| − | <li>Deactivate restriction enzymes via heat shock by incubating tube at 80°C for 20 minutes. </li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Agarose Gel Electrophoresis <i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Fragments of DNA are separated by size on the gel. This was used to visualize the results of restriction digests, particularly those done to confirm ligation or transformation.</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>TAE buffer:</p>
| + | |
| − | <ul>
| + | |
| − | <li style="margin-left: 6%;">40 mM Tris, pH 7.6</li>
| + | |
| − | <li style="margin-left: 6%;">20 mM CH₃COOH</li>
| + | |
| − | <li style="margin-left: 6%;">1 mM EDTA</li>
| + | |
| − | </ul>
| + | |
| − | <p>Agarose</p>
| + | |
| − | <p>250 mL-Erlenmeyer flask</p>
| + | |
| − | <p>RedSafe Nucleic Acid Staining Solution</p>
| + | |
| − | <p>Gel casting tray and comb</p>
| + | |
| − | <p>10X loading dye</p>
| + | |
| − | <p>DNA sample</p>
| + | |
| − | <p>Microwave</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>For a 1% gel (standard), add 0.3 g agarose to 30 mL TAE buffer in a 250 mL-Erlenmeyer flask and microwave until agarose is fully dissolved (avoid boiling for too long).</li>
| + | |
| − | <li>Allow flask to cool in fumehood until warm to the touch before adding 1.5 μL RedSafe Nucleic Acid Staining Solution. Gently swirl to mix.</li>
| + | |
| − | <li>Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel.</li>
| + | |
| − | <li>Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer.</li>
| + | |
| − | <li>Load samples of 11 μL DNA containing 1 μL loading dye.</li>
| + | |
| − | <li>Run gel at 100 V for 30 minutes or until loading dye is 1/2 way down the gel.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Ligation of DNA Inserts to Plasmid Backbones <i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Digested registry DNA or digested genetic parts from IDT were ligated to either pSB1c3 or pET29B for propagation in <i>E.coli</i> DH5ɑ or protein expression in <i>E.coli</i> BL21. Later, our parts were ligated to pSB1c3 for submission to the iGEM registry. </p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Digested vector DNA</p>
| + | |
| − | <p>Digested insert DNA</p>
| + | |
| − | <p>10X DNA ligase buffer (from New England BIolabs)</p>
| + | |
| − | <p>T4 DNA ligase (1 U/ μL) (from New England Biolabs)</p>
| + | |
| − | <p>ddH2O</p>
| + | |
| − | <p>0.2-mL PCR tubes</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>To a 0.2-mL PCR tube add:</li>
| + | |
| − | <ul>
| + | |
| − | <li style="margin-left: 6%;"><p>50 ng digested vector DNA</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>Appropriate amount of digested insert DNA to give a 3:1 molar ratio of insert:vector</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>1 μL T4 DNA ligase</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>2 μL 10X T4 DNA ligase buffer</p></li>
| + | |
| − | <li style="margin-left: 6%;"><p>ddH2O to a total volume of 20 μL </p></li>
| + | |
| − | </ul>
| + | |
| − | <li>Incubate tube at room temperature overnight.</li>
| + | |
| − | <li>Use 10 μL to transform cells, store at -20°C.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Preparation of Chemically Competent Escherichia coli Cells <i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Insert text</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Insert material 1</p>
| + | |
| − | <p>Insert material 2</p>
| + | |
| − | <p>insert material 3</p>
| + | |
| − | <p>etc.</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>Step 1</li>
| + | |
| − | <li>Step 2</li>
| + | |
| − | <li>Step 3</li>
| + | |
| − | <li>etc.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | <button class="accordion"><h3><b>Transformation of <i>Escherichia coli</i><i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Chemically competent <i>E.coli</i> DH5α was transformed with pSB1c3 or pET29b containing our genetic parts in order for that plasmid and part to be propagated. Chemically competent <i>E.coli</i> Bl21 was transformed with pSB1c3 or pET29B containing our genetic parts in order for those proteins to be expressed. </p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Competent <i>E.coli</i> aliquots (200 μL )</p>
| + | |
| − | <p>DNA for transformation</p>
| + | |
| − | <p>Luria-Bertani broth or SOC Media</p>
| + | |
| − | <p>Agar plate with appropriate antibiotic</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>Thaw 200 μL aliquot of competent E.coli DH5α cells on ice just before use.</li>
| + | |
| − | <li>Add 0.3-1 μg DNA to cells (in maximum 20 μL), flick gently to mix, and place on ice for 30 minutes.</li>
| + | |
| − | <li>Heat shock for 60-75 seconds at 42°C.</li>
| + | |
| − | <li>Place on ice for 5 minutes</li>
| + | |
| − | <li>Add 250 μL Luria-Bertani or SOC medium to aliquot of cells.</li>
| + | |
| − | <li>Incubate cells for 60 minutes at 37°C, shaking at 200 rpm for 1 hourv</li>
| + | |
| − | <li>Pellet cells in a microcentrifuge at 3500 g for 1 minute and discard supernatant.</li>
| + | |
| − | <li>Resuspend pellet in 250 μL Luria-Bertani broth.</li>
| + | |
| − | <li>Plate 50-100 μL of resuspended culture on agar plate with appropriate antibiotic and spread.</li>
| + | |
| − | <li>Incubate plates at 37°C overnight or until desired growth is observed.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | <button class="accordion"><h3> <b>Glycerol Stock Preparation of <i>Escherichia coli</i> <i class="fa fa-chevron-down"></i></b></h3></button>
| + | |
| − | <div class="panel">
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p ><b>Experimental Details and Rationale</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Glycerol stocks of transformed <i>E.coli</i> were prepared for long-term storage of the cells at -80°C.</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Materials</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <p>Overnight culture of transformed bacteria</p>
| + | |
| − | <p>Sterile 1.5-mL cryo-tubes</p>
| + | |
| − | <p>Sterile 50% glycerol</p>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <tr><td>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <ol><p>
| + | |
| − | <li>Using aseptic technique, pipette 0.5 mL of 50% sterile glycerol into a 1.5-mL cryo-tube.</li>
| + | |
| − | <li>Using aseptic technique add 0.5 mL of overnight culture.</li>
| + | |
| − | <li>Pipette up and down gently to mix.</li>
| + | |
| − | <li>Store at -80°C for up to 1 year.</li>
| + | |
| − | </p></ol>
| + | |
| − | </tr></td>
| + | |
| − |
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | </html>
| + | |
| − | }}
| + | |
| − | | + | |
| − | <html>
| + | |
| − | <head>
| + | |
| − | <style>
| + | |
| − | | + | |
| − | button.accordion.active i.fa-chevron-down {
| + | |
| − | -webkit-transform: rotate(180deg);
| + | |
| − | -moz-transform: rotate(180deg);
| + | |
| − | -o-transform: rotate(180deg);
| + | |
| − | -ms-transform: rotate(180deg);
| + | |
| − | transform: rotate(180deg);
| + | |
| − | -webkit-transition: all 0.4s ease;
| + | |
| − | -o-transition: all 0.4s ease;
| + | |
| − | transition: all 0.4s ease;
| + | |
| − | }
| + | |
| − | | + | |
| − | button.accordion {
| + | |
| − | background-color: #faece5;
| + | |
| − | color: black;
| + | |
| − | cursor: pointer;
| + | |
| − | width: 100%;
| + | |
| − | border: none;
| + | |
| − | -moz-border-radius: 4px;
| + | |
| − | -webkit-border-radius: 4px;
| + | |
| − | border-radius: 15px;
| + | |
| − | text-align: left;
| + | |
| − | outline: none;
| + | |
| − | font-size: 15px;
| + | |
| − | transition: 0.4s;
| + | |
| − | margin-top: 2%;
| + | |
| − | padding-left: 2rem;
| + | |
| − | padding-right: 6rem;
| + | |
| − | padding-top: 1rem;
| + | |
| − | padding-bottom: 0.5rem;
| + | |
| − | }
| + | |
| − | | + | |
| − | button.accordion.active, button.accordion:hover {
| + | |
| − | background-color: #fcf3ef;
| + | |
| − | }
| + | |
| − | | + | |
| − | button.accordion i.fa-chevron-down {
| + | |
| − | position: absolute;
| + | |
| − | right: 15%;
| + | |
| − | -webkit-transition: all 0.4s ease;
| + | |
| − | -o-transition: all 0.4s ease;
| + | |
| − | transition: all 0.4s ease;
| + | |
| − | }
| + | |
| − | | + | |
| − | div.panel {
| + | |
| − | padding: 1% 1%;
| + | |
| − | display: none;
| + | |
| − | background-color: white;
| + | |
| − | }
| + | |
| − | | + | |
| − | #HQ_page p {
| + | |
| − | text-align: left;
| + | |
| − | }
| + | |
| − | | + | |
| − | | + | |
| − | /*MEDIA QUERIES FOR RESIZING PURPOSES*/
| + | |
| − | @media only screen and (max-width: 70rem){
| + | |
| − | button.accordion i.fa-chevron-down {
| + | |
| − | right: 40%;
| + | |
| − | }
| + | |
| − | }
| + | |
| − | | + | |
| − | | + | |
| − | @media only screen and (max-width: 58.75rem){
| + | |
| − | | + | |
| − | button.accordion i.fa-chevron-down {
| + | |
| − | right: 24%;
| + | |
| − | }
| + | |
| − | }
| + | |
| − | | + | |
| − | @media only screen and (max-width: 44.68rem){
| + | |
| − | button.accordion i.fa-chevron-down {
| + | |
| − | right: 5%;
| + | |
| − | }
| + | |
| − | }
| + | |
| − | | + | |
| − | | + | |
| − | </style>
| + | |
| − | </head>
| + | |
| − | | + | |
| − | <script>
| + | |
| − | var acc = document.getElementsByClassName("accordion");
| + | |
| − | var i;
| + | |
| − | | + | |
| − | for (i = 0; i < acc.length; i++) {
| + | |
| − | acc[i].onclick = function(){
| + | |
| − | this.classList.toggle("active");
| + | |
| − | var panel = this.nextElementSibling;
| + | |
| − | if (panel.style.display === "block") {
| + | |
| − | panel.style.display = "none";
| + | |
| − | } else {
| + | |
| − | panel.style.display = "block";
| + | |
| − | }
| + | |
| − | }
| + | |
| − | }
| + | |
| − | </script>
| + | |
| − | | + | |
| − | </html>
| + | |
