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<br><br><p style="font-size:20px"> | <br><br><p style="font-size:20px"> | ||
<ol> | <ol> | ||
− | <li>We got RFP sequence from registry and cloned RFP gene by PCR. Then we separated cloned RFP</li> | + | <li style="font-size:16px">We got RFP sequence from registry and cloned RFP gene by PCR. Then we separated cloned RFP</li> |
− | <li>gene by agarose gel electrophoresis and purified the gene by gel slices. </li> | + | <li style="font-size:16px">gene by agarose gel electrophoresis and purified the gene by gel slices. </li> |
− | <li>We used pAK1900 as our vector. Then we used BamH I and Sac I to digest pAK1900 and purified gene for 5 hours.</li> | + | <li style="font-size:16px">We used pAK1900 as our vector. Then we used BamH I and Sac I to digest pAK1900 and purified gene for 5 hours.</li> |
− | <li>Then we used T4 DNA Ligase to ligated our vector and RFP gene at 4 ℃ for 24 hours</li> | + | <li style="font-size:16px">Then we used T4 DNA Ligase to ligated our vector and RFP gene at 4 ℃ for 24 hours</li> |
− | <li>We transferred our vector into E.coli DH5&alpha competent cell to get more vector clones. Our transferring protocol is as follows:</li> | + | <li style="font-size:16px">We transferred our vector into E.coli DH5&alpha competent cell to get more vector clones. Our transferring protocol is as follows:</li> |
<ol type="a"> | <ol type="a"> | ||
− | <li>Mix 4ul vector and 50ul competent cells in 1.5mL centrifuge tube at 0 ℃ for 30 min. </li> | + | <li style="font-size:16px">Mix 4ul vector and 50ul competent cells in 1.5mL centrifuge tube at 0 ℃ for 30 min. </li> |
− | <li>Hot shock for 90 seconds at 42℃ . </li> | + | <li style="font-size:16px">Hot shock for 90 seconds at 42℃ . </li> |
− | <li>Keep competent cell at 0℃ for 4-5 min, then add 950µL Soc media into the tube. </li> | + | <li style="font-size:16px">Keep competent cell at 0℃ for 4-5 min, then add 950µL Soc media into the tube. </li> |
− | <li>Incubate at 37 ℃ for 2 hours shaking at 220rpm</li> | + | <li style="font-size:16px">Incubate at 37 ℃ for 2 hours shaking at 220rpm</li> |
− | <li>Centrifuge at 6000rpm for 1min. After centrifugation, 900µL supernatant is discarded, then resuspended cells, spread the culture on plate with antibiotic 100µg/µL TC , incubate at 37℃ for 16 hours. </li> | + | <li style="font-size:16px">Centrifuge at 6000rpm for 1min. After centrifugation, 900µL supernatant is discarded, then resuspended cells, spread the culture on plate with antibiotic 100µg/µL TC , incubate at 37℃ for 16 hours. </li> |
− | <li>Select several colonies, purified them on another selective plate, incubate at 37℃ for 12 hours. </li> | + | <li style="font-size:16px">Select several colonies, purified them on another selective plate, incubate at 37℃ for 12 hours. </li> |
</ol> | </ol> | ||
− | <li>Incubate purified cultures with 5mL LB media and 5ul 5mg/µL TC for 5 hours. </li> | + | <li style="font-size:16px">Incubate purified cultures with 5mL LB media and 5ul 5mg/µL TC for 5 hours. </li> |
− | <li>Using Plasmid minipreparation kit to extract the vector. </li> | + | <li style="font-size:16px">Using Plasmid minipreparation kit to extract the vector. </li> |
</ol> | </ol> | ||
</p> | </p> | ||
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<div id="child8" style="display:none;"> | <div id="child8" style="display:none;"> | ||
<br><br><p style="font-size:20px"><ol> | <br><br><p style="font-size:20px"><ol> | ||
− | <li>We used Hind I, Hind II, Sal I, Hind III and Xba I, Spe I and Sal I digest pAK1900, GntR, AlkB2 for 5 hours respectively. </li> | + | <li style="font-size:16px">We used Hind I, Hind II, Sal I, Hind III and Xba I, Spe I and Sal I digest pAK1900, GntR, AlkB2 for 5 hours respectively. </li> |
− | <li>Then we used T4 DNA Ligase to ligated them at 4℃ for 24 hours. </li> | + | <li style="font-size:16px">Then we used T4 DNA Ligase to ligated them at 4℃ for 24 hours. </li> |
− | <li>Then we cloned our vector in DH5&alpha competent cells. </li> | + | <li style="font-size:16px">Then we cloned our vector in DH5&alpha competent cells. </li> |
− | <li>Sequence our gene to verify our vector. </li> | + | <li style="font-size:16px">Sequence our gene to verify our vector. </li> |
</ol></p> | </ol></p> | ||
</div> | </div> |
Latest revision as of 06:22, 1 November 2017
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