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| − | <h1><strong>Protocol</strong></h1>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="timeline-item">
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| − | <p><div class=" m-b-xs text_font" ><strong>1.Extract Arabidopsis genomic DNA</strong></div></p> | + | |
| − | <p><div class="text_font">Material:</div></p>
| + | |
| − | <ul>
| + | |
| − | <p><div class="text_font"><li>EZ-10 Spin Column Plant Genomic DNA Purification Kit</li></div></p>
| + | |
| − | <p><div class="text_font"><li>β-mercaptoethanol</li></div></p>
| + | |
| − | <p><div class="text_font"><li>RNaseA</li></div></p>
| + | |
| − | <p><div class="text_font"><li>Chloroform</li></div></p>
| + | |
| − | <p><div class="text_font"><li>Ice box and ice</li></div></p>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="timeline-item">
| + | |
| − | <div class="row">
| + | |
| − | <div class="col-xs-3 date">
| + | |
| − | </div>
| + | |
| − | <div class="col-xs-8 content">
| + | |
| − | <p><div class="m-b-xs text_font"><strong>2.PCR</strong></div></p>
| + | |
| − | <p><div class="text_font">Material:</div></p>
| + | |
| − | <ul>
| + | |
| − | <p><div class="text_font"><li>ddH20</li></div></p>
| + | |
| − | <p><div class="text_font"><li>dNTPs mix</li></div></p>
| + | |
| − | <p><div class="text_font"><li>10×Buffer</li></div></p>
| + | |
| − | <p><div class="text_font"><li>Taq polymerase</li></div></p>
| + | |
| − | <p><div class="text_font"><li>PCR Primers</li></div></p>
| + | |
| − | <p><div class="text_font"><li>Arabidopsis genomic DNA</li></div></p>
| + | |
| − | </ul>
| + | |
| − | <p><div class="text_font">Methods:</div></p>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | <p><div class="text_font">add material into a 0.2ml EP tube according to the table</div></p>
| + | |
| − | <table class="table">
| + | |
| − | <thead>
| + | |
| − | <tr>
| + | |
| − | <th>Material</th>
| + | |
| − | <th>dosage/μl</th>
| + | |
| − | </tr>
| + | |
| − | </thead>
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>dNTPs mix</td>
| + | |
| − | <td>1</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Forward primer</td>
| + | |
| − | <td>0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Reverse primer</td>
| + | |
| − | <td>0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Arabidopsis genomic DNA</td>
| + | |
| − | <td>3</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>ddH20</td>
| + | |
| − | <td>12</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Taq polymerase</td>
| + | |
| − | <td>0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Total</td>
| + | |
| − | <td>20</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <p><div class="text_font">set the PCR program </div></p>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <p><div class="text_font">For promoter cop1,phyB,pif1,gene hhl1,gene flu:</div></p>
| + | |
| − | <p><div class="text_font">94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)</div></p>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <p><div class="text_font">For pHhl1-gene hhl1:</div></p>
| + | |
| − | <p><div class="text_font">94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)</div></p>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <p><div class="text_font">Tm of each primer: </div></p>
| + | |
| − | <table class="table">
| + | |
| − | <thead>
| + | |
| − | <tr>
| + | |
| − | <th>Name</th>
| + | |
| − | <th>Tm(Forward/Reverse)(℃)</th>
| + | |
| − | <th>Tm in PCR(℃)</th>
| + | |
| − | </tr>
| + | |
| − | </thead>
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Promoter pif1</td>
| + | |
| − | <td>62.59/62.67</td>
| + | |
| − | <td>63</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Promoter cop1</td>
| + | |
| − | <td>66.77/65.46</td>
| + | |
| − | <td>66</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Promoter phyB</td>
| + | |
| − | <td>63.94/63.90</td>
| + | |
| − | <td>64</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>pHhl1-genehhl1</td>
| + | |
| − | <td>60.75/59.38</td>
| + | |
| − | <td>60</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>gene hhl1</td>
| + | |
| − | <td>60.90/59.38</td>
| + | |
| − | <td>60</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Fluorescent gene flu</td>
| + | |
| − | <td>62.42/66.90</td>
| + | |
| − | <td>65</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="timeline-item">
| + | |
| − | <div class="row">
| + | |
| − | <div class="col-xs-3 date">
| + | |
| − | </div>
| + | |
| − | <div class="col-xs-8 content">
| + | |
| − | <p><div class="m-b-xs text_font"><strong>3.digestion</strong></div></p>
| + | |
| − | <p><div class="text_font">Material:</div></p>
| + | |
| − | <ul>
| + | |
| − | <p><div class="text_font"><li>ddH20</li></div></p>
| + | |
| − | <p><div class="text_font"><li>BsaⅠBuffer</li></div></p>
| + | |
| − | <p><div class="text_font"><li>PstⅠBuffer</li></div></p>
| + | |
| − | <p><div class="text_font"><li>restriction enzyme BsaⅠ，PstⅠ，EcorⅠ</li></div></p>
| + | |
| − | <p><div class="text_font"><li>PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9</li></div></p>
| + | |
| − | <p><div class="text_font"><li>Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9</li></div></p>
| + | |
| − | </ul>
| + | |
| − | <p><div class="text_font">Method</div></p>
| + | |
| − | <ol>
| + | |
| − | <p><div class="text_font"><li>
| + | |
| − | add materials into a 1.5ml EP tube according to the table below:</div></p>
| + | |
| − | <table class="table">
| + | |
| − | <thead>
| + | |
| − | <tr>
| + | |
| − | <th>Material</th>
| + | |
| − | <th>dosage/μl</th>
| + | |
| − | </tr>
| + | |
| − | </thead>
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>PCR product(after purification)</td>
| + | |
| − | <td>17</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>restriction enzyme buffer</td>
| + | |
| − | <td>3 (/4)</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>restriction enzyme</td>
| + | |
| − | <td>1(0.5 each)</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>ddH20</td>
| + | |
| − | <td>4</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Total</td>
| + | |
| − | <td>25</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <br>
| + | |
| − | <table class="table">
| + | |
| − | <thead>
| + | |
| − | <tr>
| + | |
| − | <th>PCR product</th>
| + | |
| − | <th>cutting sites</th>
| + | |
| − | <th>restriction enzyme Buffer</th>
| + | |
| − | </tr>
| + | |
| − | </thead>
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>pHhl1-hhl1</td>
| + | |
| − | <td>BsaⅠ+EcorⅠ</td>
| + | |
| − | <td>3μlPstⅠ+1μlBsaⅠ</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>hhl1</td>
| + | |
| − | <td>PstⅠ+EcorⅠ</td>
| + | |
| − | <td>3μlPstⅠ</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>flu</td>
| + | |
| − | <td>EcorⅠ+BsaⅠ</td>
| + | |
| − | <td>3μlPstⅠ+1μlBsa1Ⅰ</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>cop1</td>
| + | |
| − | <td>BsaⅠ+PstⅠ</td>
| + | |
| − | <td>3μlPstⅠ</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>phyB</td>
| + | |
| − | <td>BsaⅠ+PstⅠ</td>
| + | |
| − | <td>3μlPstⅠ</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>pif1</td>
| + | |
| − | <td>BsaⅠ+PstⅠ</td>
| + | |
| − | <td>3μlPstⅠ</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | </li>
| + | |
| − | <p><div class="text_font"><li>Place the tube in a 37℃ incubator, stand for 1 hour.</li></div></p>
| + | |
| − | <p><div class="text_font"><li>Water bath the tube in 65℃ for 20 mins to end the digestion reaction.</li></div></p>
| + | |
| − | <ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="timeline-item">
| + | |
| − | <div class="row">
| + | |
| − | <div class="col-xs-3 date"></div>
| + | |
| − | <div class="col-xs-8 content">
| + | |
| − | <p><div class="m-b-xs text_font"><strong>4.chemical conversion</strong></div></p>
| + | |
| − | <p><div class="text_font">(1)preparation of LB Broth</div></p>
| + | |
| − | <ul>
| + | |
| − | <p><div class="text_font"><li>2g peptone</li></div></p>
| + | |
| − | <p><div class="text_font"><li>1g NaCL</li></div></p>
| + | |
| − | <p><div class="text_font"><li>0.2g glucose</li></div></p>
| + | |
| − | <p><div class="text_font"><li>1g yeast</li></div></p>
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| − | <p><div class="text_font"><li>Add water to 200ml, regulate the pH at a range of 6.8~7.2</li></div></p>
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| − | </ul>
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| − | <p><div class="text_font">Add water to 200ml, regulate the pH at a range of 6.8~7.2</div></p>
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| − | <p><div class="text_font">(2)preparation of LB Broth Medium</div></p>
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| − | <p><div class="text_font">(3)Preparation of chloromycetin solution</div></p>
| + | |
| − | <ul>
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| − | <p><div class="text_font"><li>10ml ddH20</li></div></p>
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| − | <p><div class="text_font"><li>0.25g Chloromycetin</li></div></p>
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| − | </ul>
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| − | <p><div class="text_font">Add trace amount of NaOH until the chloromycetin dissolves completely</div></p>
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| − | <p><div class="text_font">(4)add chloromycetin solution into LB broth, mix them</div></p>
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| − | <p><div class="text_font">(5)Add competence DH5α （100μl）into 10 μl plasmid，mix them gently, and ice bath</div></p>
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| − | <p><div class="text_font">(6)Heat the solution in 42℃ water bath，and ice-bath 2min (Be sure not to shake the centrifuge tube)</div></p>
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| − | <p><div class="text_font">(7)Add 890μl, 37℃ preheating LB broth, mix them gently ,shake them in the condition 37℃, 150rpm for 45min.</div></p>
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| − | <p><div class="text_font">(8)4000rpm centrifuge 5min, discard 900 μl supernatant ,suspend the 100μl sediment gentlely</div></p>
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| − | <p><div class="text_font">(9)Spread the solution on the LB broth medium evenly, after it is dried, culture it upside down in the 37 ℃ incubator for 16 hours</div></p>
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| − | <p><div class="text_font">(10)Put the centrifuge tube which including culture solution and competence DH5α in the bed temperature incubator, shaking culture it in the condition of 220rpm, 37℃ for 16 hours</div></p>
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| − | <p><div class="text_font">*THE RESUALT：</div></p>
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| − | <p><div class="text_font">(1)The escherichia coli didn't grow on the LB broth medium</div></p>
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| − | <p><div class="text_font">(2)After centrifuged, some escherichia colis can be found with a little amount in the bottom of the centrifuge tubes</div></p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="col-xs-3 date"></div>
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| − | <div class="col-xs-8 content">
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| − | <p><div class="m-b-xs text_font"><strong>5.plasmid extraction</strong></div></p>
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| − | <p><div class="text_font">Experiment macterial: the kit for plasmid extraction</div></p>
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| − | <p><div class="text_font">Experiment apparatus: centrifugal machine, water bath, incubator</div></p>
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| − | <p><div class="text_font">Experiment preparation: 60 ℃ water bath， 37 ℃ incubator</div></p>
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| − | <p><div class="text_font">(1)centrifuge the bacterial liqiud ,12000rpm 1min. Discard the supernatant.</div></p>
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| − | <p><div class="text_font">(2)According to the kit for extraction, extract the plasmid DNA</div></p>
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| − | <p><div class="text_font">(3)Preserve the plasmid in 4℃ refrigerator</div></p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="timeline-item">
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| − | <div class="col-xs-3 date"></div>
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| − | <div class="col-xs-8 content">
| + | |
| − | <p><div class="m-b-xs text_font"><strong>6.extraction of RNA</strong></div></p>
| + | |
| − | <p><div class="text_font">Experiment macterial：the kit for RNA fractionation and purification</div></p>
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| − | <p><div class="text_font">Experiment steps: refer to protocol</div></p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="col-xs-3 date"></div>
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| − | <div class="col-xs-8 content">
| + | |
| − | <p><div class="m-b-xs text_font"><strong>7.RT-PCR</strong></div></p>
| + | |
| − | <p><div class="text_font">Experiment macterial：M-MuLV</div></p>
| + | |
| − | <p><div class="text_font">(1)preparation for RT-PCR</div></p>
| + | |
| − | <ul>
| + | |
| − | <p><div class="text_font"><li>Buffer 5μl</li></div></p>
| + | |
| − | <p><div class="text_font"><li>Forward Primer 2μl</li></div></p>
| + | |
| − | <p><div class="text_font"><li>Reverse primer 2μl</li></div></p>
| + | |
| − | <p><div class="text_font"><li>RT reverse transcription 0.5μl</li></div></p>
| + | |
| − | <p><div class="text_font"><li>Taq enzyme 0.5μl</li></div></p>
| + | |
| − | <p><div class="text_font"><li>RNA 20μl</li></div></p>
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| − | <p><div class="text_font"><li>ddH2O 19.5μl</li></div></p>
| + | |
| − | </ul>
| + | |
| − | <p><div class="text_font">(2)RT-PCR program</div></p>
| + | |
| − | <p><div class="text_font">45℃×30min+94℃×5min+（94℃×30s+60.5℃×30s+72℃×1min）×40+72℃×10min</div></p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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