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<li>gene by agarose gel electrophoresis and purified the gene by gel slices. </li> | <li>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>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 &# | + | <li>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>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 &# | + | <li>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>Hot shock for 90 seconds at 42℃ . </li> |
− | <li>Keep competent cell at 0&# | + | <li>Keep competent cell at 0℃ for 4-5 min, then add 950µL Soc media into the tube. </li> |
− | <li>Incubate at 37 &# | + | <li>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&# | + | <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>Select several colonies, purified them on another selective plate, incubate at 37&# | + | <li>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>Incubate purified cultures with 5mL LB media and 5ul 5mg/µL TC for 5 hours. </li> | ||
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<p><ol> | <p><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>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&# | + | <li>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>Then we cloned our vector in DH5&alpha competent cells. </li> | ||
<li>Sequence our gene to verify our vector. </li> | <li>Sequence our gene to verify our vector. </li> |
Revision as of 07:19, 16 October 2017