http://2009.igem.org/wiki/index.php?title=Team:KU_Seoul/Experiment_Dairy&feed=atom&action=historyTeam:KU Seoul/Experiment Dairy - Revision history2024-03-28T20:22:21ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:KU_Seoul/Experiment_Dairy&diff=139860&oldid=prevBys131: /* Experiment Dairy */2009-10-21T07:54:19Z<p><span class="autocomment">Experiment Dairy</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=== Experiment <del class="diffchange diffchange-inline">Dairy </del>===</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=== Experiment <ins class="diffchange diffchange-inline">Diary </ins>===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090914</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090914</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Streaking of ''E. coli'' XL-1 Blue</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Streaking of ''E. coli'' XL-1 Blue</div></td></tr>
</table>Bys131http://2009.igem.org/wiki/index.php?title=Team:KU_Seoul/Experiment_Dairy&diff=127528&oldid=prevRoh329 at 12:11, 20 October 20092009-10-20T12:11:43Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Incubation for 60min at 37℃ and centrifugation at 8,000rpm for 1min</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Incubation for 60min at 37℃ and centrifugation at 8,000rpm for 1min</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Resuspension to 1ml reaction mixture ( 100mM AAP in 0.1M Tris buffer pH 9.0) and incubation for 10min at 37℃</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Resuspension to 1ml reaction mixture ( 100mM AAP in 0.1M Tris buffer pH 9.0) and incubation for 10min at 37℃</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>##Stopped with the addition of 2 mL of 1 % (vol/vol) o-cresol and 0.2 mL of 0.2 % (wt/vol) <del class="diffchange diffchange-inline">CuSO4 </del>in 1.6 % (vol/vol) NH<Sub>4</Sub>OH. After 10 min of incubation at room temperature</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>##Stopped with the addition of 2 mL of 1 % (vol/vol) o-cresol and 0.2 mL of 0.2 % (wt/vol) <ins class="diffchange diffchange-inline">CuSO<Sub>4</Sub> </ins>in 1.6 % (vol/vol) NH<Sub>4</Sub>OH. After 10 min of incubation at room temperature</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##The amount of p-aminophenol was determined by a spectrophotometer (615 nm)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##The amount of p-aminophenol was determined by a spectrophotometer (615 nm)</div></td></tr>
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</table>Roh329http://2009.igem.org/wiki/index.php?title=Team:KU_Seoul/Experiment_Dairy&diff=127517&oldid=prevRoh329 at 12:10, 20 October 20092009-10-20T12:10:45Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Streaking of ''E. coli'' XL-1 Blue</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Streaking of ''E. coli'' XL-1 Blue</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Preparation of LB plate (containing 100μg/ml ampicillin, 12.5μg/ml tetracycline, 50μg/ml kanamycin and 25μg/ml chloramphenicol of final concentration, respectively) & dry in 37℃ incubator for 12 hours</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Preparation of LB plate (containing 100μg/ml ampicillin, 12.5μg/ml tetracycline, 50μg/ml kanamycin and 25μg/ml chloramphenicol of final concentration, respectively) & dry in 37℃ incubator for 12 hours</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Inoculation of E. coli DH5a to 5ml LB broth for preparation of competent cell</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Inoculation of <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>DH5a to 5ml LB broth for preparation of competent cell</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Design of cloning primers & ordering the primers</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Design of cloning primers & ordering the primers</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090915</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090915</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Inoculation of E. coli XL-1 Blue to 2ml LB broth for genomic DNA extraction</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Inoculation of <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>XL-1 Blue to 2ml LB broth for genomic DNA extraction</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Preparation of E. coli DH5a competent cell (based on BSGC protocol)</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Preparation of <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>DH5a competent cell (based on BSGC protocol)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090917</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090917</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Transformation for amplification of BioBrick parts (BBa_E0044, BBa_E1010, pSB3C5 and pSB3T5) (based on BSGC protocol)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Transformation for amplification of BioBrick parts (BBa_E0044, BBa_E1010, pSB3C5 and pSB3T5) (based on BSGC protocol)</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Genomic DNA extraction of E. coli XL-1 Blue by AccuPrep® Genomic DNA Extraction Kit</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Genomic DNA extraction of <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>XL-1 Blue by AccuPrep® Genomic DNA Extraction Kit</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090918</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090918</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Inoculation of colonies to 2ml LBAmp(BBa_E0044), LBKan(BBa_E1010), LBCm(pSB3C5) and LBTet(pSB3T5) broth</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Inoculation of colonies to 2ml LBAmp(BBa_E0044), LBKan(BBa_E1010), LBCm(pSB3C5) and LBTet(pSB3T5) broth</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Cloning of Pars-gfp-Pznt-rfp to pSB3C5 & PyodA-AMD to pSB3T5</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Cloning of Pars-gfp-Pznt-rfp to pSB3C5 & PyodA-AMD to pSB3T5</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Annealing & Transformation : 2ul insert + 2ul pSB3 vector at room temperature for 30min</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Annealing & Transformation : 2ul insert + 2ul pSB3 vector at room temperature for 30min</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>##Transformation to E. coli DH5a</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>##Transformation to <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>DH5a</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Results >> vector only : 2, vector + INS : 4</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Results >> vector only : 2, vector + INS : 4</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090925-27, 090929~091001</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090925-27, 090929~091001</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*091005~091006</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*091005~091006</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Sequencing by Macrogen</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Sequencing by Macrogen</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Transformation to E. coli DH5a</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Transformation to <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>DH5a</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*091010~091020</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*091010~091020</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Procedure for detecting <del class="diffchange diffchange-inline">Zn2</del>+ and <del class="diffchange diffchange-inline">AsO3</del>- & Construction of calibration curve for red fluorescence</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Procedure for detecting <ins class="diffchange diffchange-inline">Zn<Sup>2</ins>+<ins class="diffchange diffchange-inline"></Sup> </ins>and <ins class="diffchange diffchange-inline">AsO<Sup>3</ins>-<ins class="diffchange diffchange-inline"></Sup> </ins>& Construction of calibration curve for red fluorescence</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>##E. coli DH5a harboring pSB3C5/Pars-gfp-Pznt-rfp >> inoculation to 5ml LBC broth and incubation at 37℃ for 12h</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>##<ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>DH5a harboring pSB3C5/Pars-gfp-Pznt-rfp >> inoculation to 5ml LBC broth and incubation at 37℃ for 12h</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>##Addition of various concentration of <del class="diffchange diffchange-inline">Zn2</del>+ (0, 0.25, 0.5, 1, 1.5, 2mM) & <del class="diffchange diffchange-inline">AsO3</del>- (0, 0.0625, 0.125, 0.25, 0.5, 0.75, 1mM) to 1ml incubated broth</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>##Addition of various concentration of <ins class="diffchange diffchange-inline">Zn<Sup>2</ins>+<ins class="diffchange diffchange-inline"></Sup> </ins>(0, 0.25, 0.5, 1, 1.5, 2mM) & <ins class="diffchange diffchange-inline">AsO<Sup>3</ins>-<ins class="diffchange diffchange-inline"></Sup> </ins>(0, 0.0625, 0.125, 0.25, 0.5, 0.75, 1mM) to 1ml incubated broth</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Incubation for 60min at 37℃</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Incubation for 60min at 37℃</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Detection by Multilabel Plate Reader (FL/LU) (Perkin Elmer) and Microplate Spectrophotometer (Bio-Tek)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Detection by Multilabel Plate Reader (FL/LU) (Perkin Elmer) and Microplate Spectrophotometer (Bio-Tek)</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Procedure for detecting <del class="diffchange diffchange-inline">Cd2</del>+ & Construction of calibration curve</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Procedure for detecting <ins class="diffchange diffchange-inline">Cd<Sup>2</ins>+<ins class="diffchange diffchange-inline"></Sup> </ins>& Construction of calibration curve</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>##E. coli DH5a harboring pSB3T5/PyodA-AMD >> inoculation to 5ml LBT broth and incubation at 37℃ for 12h</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>##<ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>DH5a harboring pSB3T5/PyodA-AMD >> inoculation to 5ml LBT broth and incubation at 37℃ for 12h</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>##Addition of various concentration of <del class="diffchange diffchange-inline">Cd2</del>+ (0, 0.05, 0.1, 0.2, 0.4 and 0.8mM) to 1ml incubated broth and storage at 4℃</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>##Addition of various concentration of <ins class="diffchange diffchange-inline">Cd<Sup>2</ins>+<ins class="diffchange diffchange-inline"></Sup> </ins>(0, 0.05, 0.1, 0.2, 0.4 and 0.8mM) to 1ml incubated broth and storage at 4℃</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Incubation for 60min at 37℃ and centrifugation at 8,000rpm for 1min</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Incubation for 60min at 37℃ and centrifugation at 8,000rpm for 1min</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Resuspension to 1ml reaction mixture ( 100mM AAP in 0.1M Tris buffer pH 9.0) and incubation for 10min at 37℃</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##Resuspension to 1ml reaction mixture ( 100mM AAP in 0.1M Tris buffer pH 9.0) and incubation for 10min at 37℃</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>##Stopped with the addition of 2 mL of 1 % (vol/vol) o-cresol and 0.2 mL of 0.2 % (wt/vol) CuSO4 in 1.6 % (vol/vol) <del class="diffchange diffchange-inline">NH4OH</del>. After 10 min of incubation at room temperature</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>##Stopped with the addition of 2 mL of 1 % (vol/vol) o-cresol and 0.2 mL of 0.2 % (wt/vol) CuSO4 in 1.6 % (vol/vol) <ins class="diffchange diffchange-inline">NH<Sub>4</Sub>OH</ins>. After 10 min of incubation at room temperature</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##The amount of p-aminophenol was determined by a spectrophotometer (615 nm)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>##The amount of p-aminophenol was determined by a spectrophotometer (615 nm)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr>
</table>Roh329http://2009.igem.org/wiki/index.php?title=Team:KU_Seoul/Experiment_Dairy&diff=127479&oldid=prevRoh329 at 12:06, 20 October 20092009-10-20T12:06:27Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Experiment Dairy ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Experiment Dairy ===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090914</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*090914</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Streaking of E. coli XL-1 Blue</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Streaking of <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>XL-1 Blue</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Preparation of LB plate (containing 100μg/ml ampicillin, 12.5μg/ml tetracycline, 50μg/ml kanamycin and 25μg/ml chloramphenicol of final concentration, respectively) & dry in 37℃ incubator for 12 hours</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Preparation of LB plate (containing 100μg/ml ampicillin, 12.5μg/ml tetracycline, 50μg/ml kanamycin and 25μg/ml chloramphenicol of final concentration, respectively) & dry in 37℃ incubator for 12 hours</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Inoculation of E. coli DH5a to 5ml LB broth for preparation of competent cell</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Inoculation of E. coli DH5a to 5ml LB broth for preparation of competent cell</div></td></tr>
</table>Roh329http://2009.igem.org/wiki/index.php?title=Team:KU_Seoul/Experiment_Dairy&diff=127443&oldid=prevRoh329: New page: {{:Team:KU_Seoul/mainframe}} <html> <head> <style> #bodyContent {background-color: #7C001A;} #content {background-color: #7C001A;} #footer-box {background-color: #CCCCCC;} P {color: #0000...2009-10-20T12:02:28Z<p>New page: {{:Team:KU_Seoul/mainframe}} <html> <head> <style> #bodyContent {background-color: #7C001A;} #content {background-color: #7C001A;} #footer-box {background-color: #CCCCCC;} P {color: #0000...</p>
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=== Experiment Dairy ===<br />
*090914<br />
#Streaking of E. coli XL-1 Blue<br />
#Preparation of LB plate (containing 100μg/ml ampicillin, 12.5μg/ml tetracycline, 50μg/ml kanamycin and 25μg/ml chloramphenicol of final concentration, respectively) & dry in 37℃ incubator for 12 hours<br />
#Inoculation of E. coli DH5a to 5ml LB broth for preparation of competent cell<br />
#Design of cloning primers & ordering the primers<br />
*090915<br />
#Inoculation of E. coli XL-1 Blue to 2ml LB broth for genomic DNA extraction<br />
#Preparation of E. coli DH5a competent cell (based on BSGC protocol)<br />
*090917<br />
#Transformation for amplification of BioBrick parts (BBa_E0044, BBa_E1010, pSB3C5 and pSB3T5) (based on BSGC protocol)<br />
#Genomic DNA extraction of E. coli XL-1 Blue by AccuPrep® Genomic DNA Extraction Kit<br />
*090918<br />
#Inoculation of colonies to 2ml LBAmp(BBa_E0044), LBKan(BBa_E1010), LBCm(pSB3C5) and LBTet(pSB3T5) broth<br />
<br />
<br />
*090919<br />
#Plasmid extraction of each part by LaboPass™ Mini Plasmid DNA Purification Kit<br />
#0.8% agarose gel electrophoresis (Figure 1)<br />
[[Image:KU_Seoul_1.jpeg]]<br />
<br />
*090921<br />
#PCR of BioBrick Parts<br />
::General PCR condition : 94℃(3’)-[94℃(1’)-53℃(1’)-72℃(3’)]30-72℃(5’)-4℃ <br />
{| style="color:green; background-color:#ffffcc;" cellpadding="3" cellspacing="0" border="1"<br />
! PCR mixture <br />
! volume (μl)<br />
|-<br />
|Template (mini prep. samples of plasmid : aryl acylamidase, rfp, gfp and PyodA) ||align="center"| 1<br />
|-<br />
|2.5mM dNTP ||align="center"| 4<br />
|-<br />
|forward-primer (10pmol/μl) ||align="center"| 2<br />
|-<br />
|reverse-primer (10pmol/μl) || align="center"| 2<br />
|-<br />
|10x Synergy™ buffer || align="center"| 5<br />
|-<br />
|Synergy™ DNA polymerase [Gene Ctraft Germany] (10U/μl) || align="center"| 0.5<br />
|-<br />
|D.W. || align="center"| 35.5<br />
|}<br />
::Hot start PCR condition : 95℃(15’)-[95℃(1’)-53℃(1’)-72℃(3’)]30-72℃(10’)-4℃<br />
{| style="color:green; background-color:#ffffcc;" cellpadding="3" cellspacing="0" border="1"<br />
! PCR mixture <br />
! volume (μl)<br />
|-<br />
|Template [mini prep. samples of plasmid : pSB3C5 and pSB3T5] ||align="center"| 1<br />
|-<br />
|2.5mM dNTP ||align="center"| 4<br />
|-<br />
|forward-primer (10pmol/μl) ||align="center"| 2<br />
|-<br />
|reverse-primer (10pmol/μl) || align="center"| 2<br />
|-<br />
|10x Synergy™ buffer || align="center"| 5<br />
|-<br />
|5x Q-solution || align="center"| 10<br />
|-<br />
|Hot start taq™ (Qiagen) (5U/μl) || align="center"| 0.05<br />
|-<br />
|D.W. || align="center"| 25.5<br />
|}<br />
:2. Result (Figure 2)<br />
[[Image:KU_Seoul_2.jpg]]<br />
*090922<br />
#Part linkage PCR<br />
::Linkage PCR condition : 94℃(3’)-[94℃(1’)-53℃(1’)-72℃(3’)]30-72℃(5’)-4℃<br />
{| style="color:green; background-color:#ffffcc;" cellpadding="3" cellspacing="0" border="1"<br />
! PCR mixture <br />
! volume (μl)<br />
|-<br />
|Template [PCR products of each part diluted 10-fold]] ||align="center"| 1<br />
|-<br />
|2.5mM dNTP ||align="center"| 4<br />
|-<br />
|forward-primer (10pmol/μl) ||align="center"| 2<br />
|-<br />
|reverse-primer (10pmol/μl) || align="center"| 2<br />
|-<br />
|10x Synergy™ buffer || align="center"| 5<br />
|-<br />
|Synergy™ DNA polymerase [Gene Ctraft Germany] (10U/μl) || align="center"| 0.05<br />
|-<br />
|D.W. || align="center"| 35.5<br />
|}<br />
:2. Result (Figure 3)<br />
[[Image:KU_Seoul_3.jpg]]<br />
*090923<br />
:1.Cloning of gfp-rfp to pSB3C5 by Infusion Assembly<br />
::1) Clean-up of each PCR products by LaboPass™ Gel and PCR Clean-up Kit – elution : 43ul<br />
::2) DpnI (10U/μl) reaction of pSB3C5 and pSB3T5 PCR product at 37℃ for 3-h and clean-up – elution : 43ul<br />
::3) T4 DNA polymerase reaction of purified PCR products : Incubation at room temperature for 30min<br />
{| style="color:green; background-color:#ffffcc;" cellpadding="3" cellspacing="0" border="1"<br />
! Reaction mixture<br />
! volume (μl)<br />
|-<br />
|Purified PCR product ||align="center"| 43<br />
|-<br />
|BSA (NEB) (10mg/ml) ||align="center"| 1<br />
|-<br />
|10x NEB buffer 2 ||align="center"| 5<br />
|-<br />
|T4 DNA polymerase (0.6U/μl) || align="center"| 1<br />
|}<br />
::4) Clean-up – eleution : 40ul<br />
::5) 0.8% agarose gel electrophoresis<br />
<br />
*090923~090924<br />
#Cloning of Pars-gfp-Pznt-rfp to pSB3C5 & PyodA-AMD to pSB3T5<br />
##Annealing & Transformation : 2ul insert + 2ul pSB3 vector at room temperature for 30min<br />
##Transformation to E. coli DH5a<br />
##Results >> vector only : 2, vector + INS : 4<br />
*090925-27, 090929~091001<br />
:1. Cloning results<br />
{| style="color:green; background-color:#ffffcc;" cellpadding="3" cellspacing="0" border="1"<br />
! Plate<br />
! Number of colonies<br />
! Plasmid prep.<br />
|-<br />
|pSB3C5 vector || align="center"|2 ||align="center"| 1<br />
|-<br />
|pSB3C5 + Pars-gfp-Pznt-rfp || align="center"|5 ||align="center"| 1/3<br />
|-<br />
|pSB3T5 vector || align="center"|3 ||align="center"| 1<br />
|-<br />
|pSB3T5 + PyodA-AMD || align="center"|14 ||align="center"| 5/6<br />
|}<br />
:2. Plasmid mini prep. & 0.8% agarose gel electrophoresis (Figure 4)<br />
[[Image:KU_Seoul_4.jpg]]<br />
*091005~091006<br />
#Sequencing by Macrogen<br />
#Transformation to E. coli DH5a<br />
*091010~091020<br />
#Procedure for detecting Zn2+ and AsO3- & Construction of calibration curve for red fluorescence<br />
##E. coli DH5a harboring pSB3C5/Pars-gfp-Pznt-rfp >> inoculation to 5ml LBC broth and incubation at 37℃ for 12h<br />
##Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃<br />
##Addition of various concentration of Zn2+ (0, 0.25, 0.5, 1, 1.5, 2mM) & AsO3- (0, 0.0625, 0.125, 0.25, 0.5, 0.75, 1mM) to 1ml incubated broth<br />
##Incubation for 60min at 37℃<br />
##Detection by Multilabel Plate Reader (FL/LU) (Perkin Elmer) and Microplate Spectrophotometer (Bio-Tek)<br />
#Procedure for detecting Cd2+ & Construction of calibration curve<br />
##E. coli DH5a harboring pSB3T5/PyodA-AMD >> inoculation to 5ml LBT broth and incubation at 37℃ for 12h<br />
##Inoculation 50ml LBC and incubation to OD600 ~0.5 at 37℃<br />
##Addition of various concentration of Cd2+ (0, 0.05, 0.1, 0.2, 0.4 and 0.8mM) to 1ml incubated broth and storage at 4℃<br />
##Incubation for 60min at 37℃ and centrifugation at 8,000rpm for 1min<br />
##Resuspension to 1ml reaction mixture ( 100mM AAP in 0.1M Tris buffer pH 9.0) and incubation for 10min at 37℃<br />
##Stopped with the addition of 2 mL of 1 % (vol/vol) o-cresol and 0.2 mL of 0.2 % (wt/vol) CuSO4 in 1.6 % (vol/vol) NH4OH. After 10 min of incubation at room temperature<br />
##The amount of p-aminophenol was determined by a spectrophotometer (615 nm)<br />
|}</div>Roh329