|
|
| (127 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>Chassis</h2>
| + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td colspan="2">
| + | |
| − | <h3><b>Rehydration of Registry DNA</b></h3>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <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>
| + | |
| − | | + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td colspan="2">
| + | |
| − | <h3><b>Rehydration of IDT Synthesized DNA</b></h3>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <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>
| + | |
| − | <p><b>Protocol</b></p>
| + | |
| − | </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>
| + | |
| − | | + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td colspan="2">
| + | |
| − | <a name="plasmid_miniprep"><h3><b>Plasmid MiniPrep from <i>Escherichia coli</i></b></h3></a>
| + | |
| − | </td></tr>
| + | |
| − | | + | |
| − | <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 restriction digest, 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>
| + | |
| − | | + | |
| − | | + | |
| − | <table border="0">
| + | |
| − | | + | |
| − | <tr><td colspan = "2">
| + | |
| − | <h3><b>Restriction Digest</b></h3>
| + | |
| − | </tr></td>
| + | |
| − | | + | |
| − | <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>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | </html>
| + | |
| − | }}
| + | |
| − | | + | |
| − | <html>
| + | |
| − | <head>
| + | |
| − | <style>
| + | |
| − | #HQ_page p {
| + | |
| − | text-align: left;
| + | |
| − | }
| + | |
| − | h3{
| + | |
| − | text-align:center;
| + | |
| − | }
| + | |
| − | | + | |
| − | </style>
| + | |
| − | </head>
| + | |
| − | </html>
| + | |
