http://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&feed=atom&action=historyTeam:NRP-UEA-Norwich/Week5 - Revision history2024-03-28T10:52:42ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=226313&oldid=prevKhadijaouadi at 00:16, 27 September 20122012-09-27T00:16:09Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Week 5=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Week 5=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Again, this week was very much centered around lab work and in producing our first BioBricks. The PCR reactions gave us a <del class="diffchange diffchange-inline">god </del>starting point and we made great progress that was rewarded at the end <del class="diffchange diffchange-inline">pf </del>the <del class="diffchange diffchange-inline">weel: </del>we successfully ligated the BM and MB inserts into the plasmid backbone. We had our first <del class="diffchange diffchange-inline">bricks</del>! </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Again, this week was very much centered around lab work and in producing our first BioBricks. The PCR reactions gave us a <ins class="diffchange diffchange-inline">good </ins>starting point and we made great progress that was rewarded at the end <ins class="diffchange diffchange-inline">of </ins>the <ins class="diffchange diffchange-inline">week where </ins>we successfully ligated the BM and MB inserts into the plasmid backbone. We had our first <ins class="diffchange diffchange-inline">BioBricks</ins>! </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also began our growth study to characterise PyeaR-GFP (BBa_K381001). This has been very much a hectic week but has been totally worth while, seeing the results.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also began our growth study to characterise PyeaR-GFP (BBa_K381001). This has been very much a hectic week but has been totally worth while, seeing the results.</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>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Russell calculated the expected lengths of the plasmids containing RFP, eCFP and AraC promoter BioBricks in pCB1C3 and found that they ranged from 3.9kbp (RFP) to 2.3kbp (AraC). However the gel showed the plasmid sizes ranging from 2.5kbp (RFP and AraC2) to 1.5kbp (eCFP and AraC1). A stark difference from we had expected. We decided to carry out two resriction digests of the plasmids; one to linearise them and one to cut out the insert, the other one in order to validate the true size of the plasmid and the size of the desired insert. We then nano-dropped the <del class="diffchange diffchange-inline">smaples </del>and found that only a very small amount of DNA was present in the solutions. We expected to see DNA levels in the 100s of ng/μl however the results we gained were:</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Russell calculated the expected lengths of the plasmids containing RFP, eCFP and AraC promoter BioBricks in pCB1C3 and found that they ranged from 3.9kbp (RFP) to 2.3kbp (AraC). However the gel showed the plasmid sizes ranging from 2.5kbp (RFP and AraC2) to 1.5kbp (eCFP and AraC1). A stark difference from we had expected. We decided to carry out two resriction digests of the plasmids; one to linearise them and one to cut out the insert, the other one in order to validate the true size of the plasmid and the size of the desired insert. We then nano-dropped the <ins class="diffchange diffchange-inline">samples </ins>and found that only a very small amount of DNA was present in the solutions. We expected to see DNA levels in the 100s of ng/μl however the results we gained were:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Khadijaouadihttp://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=225967&oldid=prevYuenYeeLo: /* Labs */2012-09-27T00:08:56Z<p><span class="autocomment">Labs</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Rachel checked the development of the samples of transformed bacteria and found that some had grown but didn't quiet show as much growth as expected; they were left to incubate for a few more hours to grow before inoculation.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Rachel checked the development of the samples of transformed bacteria and found that some had grown but didn't quiet show as much growth as expected; they were left to incubate for a few more hours to grow before inoculation.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Russell carried out a restriction digest of each isolated plasmid, of which we hoped that they hopefully contained the RFP, eCFP or AraC BioBricks. He performed single digests with just EcoRI to linerarise the plasmid and double digests with EcoRI + PstI to cut out the insert. These digested plasmids will then be run on an agarose gel in order to validate the DNA, due to the unusual results previously. If the gel showed fragments of expected sizes then the BioBricks transformed into E. coli by Rachel will be used in the ligation of reporters to the hybrid promoter and to carry out quantitative experiments.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Russell carried out a restriction digest of each isolated plasmid, of which we hoped that they hopefully contained the RFP, eCFP or AraC BioBricks. He performed single digests with just EcoRI to linerarise the plasmid and double digests with EcoRI + PstI to cut out the insert. These digested plasmids will then be run on an agarose gel in order to validate the DNA, due to the unusual results previously. If the gel showed fragments of expected sizes then the BioBricks transformed into <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>by Rachel will be used in the ligation of reporters to the hybrid promoter and to carry out quantitative experiments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A plan was made to characterise PyeaR + GFP (BBa_K1001) and discussed between Lukas, Rebecca and Joy. We plan to grow up PyeaR + GFP in cultures, reading the absorbance at 660nm of the samples regularly (hourly). The inoculation will be made directly from the plate. The pilot study will last for 6 hours and the full study, if the pilot study goes according to plan, will be 12 hours. To generate a comparison of PyeaR + GFP containing cells against non transformed cells, Bioline competent cells were plated and grown for next day use. The final studies and results can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments#A_study_of_growth_of_PyeaR_in_different_concentrations_of_potassium_nitrate here].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A plan was made to characterise PyeaR + GFP (BBa_K1001) and discussed between Lukas, Rebecca and Joy. We plan to grow up PyeaR + GFP in cultures, reading the absorbance at 660nm of the samples regularly (hourly). The inoculation will be made directly from the plate. The pilot study will last for 6 hours and the full study, if the pilot study goes according to plan, will be 12 hours. To generate a comparison of PyeaR + GFP containing cells against non transformed cells, Bioline competent cells were plated and grown for next day use. The final studies and results can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments#A_study_of_growth_of_PyeaR_in_different_concentrations_of_potassium_nitrate here].</div></td></tr>
</table>YuenYeeLohttp://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=225127&oldid=prevKhadijaouadi at 23:52, 26 September 20122012-09-26T23:52:14Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 23:52, 26 September 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Week 5=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Week 5=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Again, this week was very much centered around lab work and in producing our first <del class="diffchange diffchange-inline">Biobricks</del>. The PCR reactions gave us a god starting point and we made great progress that was rewarded at the end pf the weel: we successfully ligated the BM and MB inserts into the plasmid backbone. We had our first bricks! </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Again, this week was very much centered around lab work and in producing our first <ins class="diffchange diffchange-inline">BioBricks</ins>. The PCR reactions gave us a god starting point and we made great progress that was rewarded at the end pf the weel: we successfully ligated the BM and MB inserts into the plasmid backbone. We had our first bricks! </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also began our growth study to characterise PyeaR-GFP (BBa_K381001). This has been very much a hectic week but has been totally worth while, seeing the results.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also began our growth study to characterise PyeaR-GFP (BBa_K381001). This has been very much a hectic week but has been totally worth while, seeing the results.</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>
<tr><td colspan="2" class="diff-lineno">Line 17:</td>
<td colspan="2" class="diff-lineno">Line 17:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Russel+Rachel_gel.png | thumb | '' '''Figure 1.''' Gel electrophoresis of plasmid isolations of various fluorescent BioBricks (RFP, eCFP and AraC promoter)'']]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Russel+Rachel_gel.png | thumb | '' '''Figure 1.''' Gel electrophoresis of plasmid isolations of various fluorescent BioBricks (RFP, eCFP and AraC promoter)'']]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Russell and Rachel ran their isolated plasmids containing RFP, eCFP and AraC promoter <del class="diffchange diffchange-inline">Biobricks </del>on an agarose gel and found that the bands found were not in-keeping with the size they should be. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not, resulting in the abnormal results and it was concluded that further experiments were needed (''Figure 1.''). Further validation experiments were discussed.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Russell and Rachel ran their isolated plasmids containing RFP, eCFP and AraC promoter <ins class="diffchange diffchange-inline">BioBricks </ins>on an agarose gel and found that the bands found were not in-keeping with the size they should be. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not, resulting in the abnormal results and it was concluded that further experiments were needed (''Figure 1.''). Further validation experiments were discussed.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A gel of the digested PCR products was rerun following the previous Friday's results. Again the gel was unclear so we decided to repeat the PCR restriction digest.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A gel of the digested PCR products was rerun following the previous Friday's results. Again the gel was unclear so we decided to repeat the PCR restriction digest.</div></td></tr>
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<td colspan="2" class="diff-lineno">Line 32:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Russell calculated the expected lengths of the plasmids containing RFP, eCFP and AraC promoter <del class="diffchange diffchange-inline">Biobricks </del>in pCB1C3 and found that they ranged from 3.9kbp (RFP) to 2.3kbp (AraC). However the gel showed the plasmid sizes ranging from 2.5kbp (RFP and AraC2) to 1.5kbp (eCFP and AraC1). A stark difference from we had expected. We decided to carry out two resriction digests of the plasmids; one to linearise them and one to cut out the insert, the other one in order to validate the true size of the plasmid and the size of the desired insert. We then nano-dropped the smaples and found that only a very small amount of DNA was present in the solutions. We expected to see DNA levels in the 100s of ng/μl however the results we gained were:</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Russell calculated the expected lengths of the plasmids containing RFP, eCFP and AraC promoter <ins class="diffchange diffchange-inline">BioBricks </ins>in pCB1C3 and found that they ranged from 3.9kbp (RFP) to 2.3kbp (AraC). However the gel showed the plasmid sizes ranging from 2.5kbp (RFP and AraC2) to 1.5kbp (eCFP and AraC1). A stark difference from we had expected. We decided to carry out two resriction digests of the plasmids; one to linearise them and one to cut out the insert, the other one in order to validate the true size of the plasmid and the size of the desired insert. We then nano-dropped the smaples and found that only a very small amount of DNA was present in the solutions. We expected to see DNA levels in the 100s of ng/μl however the results we gained were:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 44:</td>
<td colspan="2" class="diff-lineno">Line 44:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Despite not being in the 100s of ng/μl as desired, we decided that we could use this DNA to carry out future restriction digests and gel electrophoresis in order to validate. We then isolate DNA from the ''E. coli'', that was to be transformed by Rachel for the future restriction digest/ligation, to produce new <del class="diffchange diffchange-inline">biobricks</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Despite not being in the 100s of ng/μl as desired, we decided that we could use this DNA to carry out future restriction digests and gel electrophoresis in order to validate. We then isolate DNA from the ''E. coli'', that was to be transformed by Rachel for the future restriction digest/ligation, to produce new <ins class="diffchange diffchange-inline">BioBricks</ins>.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Rachel transformed fresh "E. coli" with the original <del class="diffchange diffchange-inline">biobricks </del>in order to produce a new stock of DNA, as we had left gaps between using the bacteria and DNA previously, which could account for the very low levels of DNA we had experienced in the nanodrops.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Rachel transformed fresh "E. coli" with the original <ins class="diffchange diffchange-inline">BioBricks </ins>in order to produce a new stock of DNA, as we had left gaps between using the bacteria and DNA previously, which could account for the very low levels of DNA we had experienced in the nanodrops.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
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<td colspan="2" class="diff-lineno">Line 55:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Rachel checked the development of the samples of transformed bacteria and found that some had grown but didn't quiet show as much growth as expected; they were left to incubate for a few more hours to grow before inoculation.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Rachel checked the development of the samples of transformed bacteria and found that some had grown but didn't quiet show as much growth as expected; they were left to incubate for a few more hours to grow before inoculation.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Russell carried out a restriction digest of each isolated plasmid, of which we hoped that they hopefully contained the RFP, eCFP or AraC BioBricks. He performed single digests with just EcoRI to linerarise the plasmid and double digests with EcoRI + PstI to cut out the insert. These digested plasmids will then be run on an agarose gel in order to validate the DNA, due to the unusual results previously. If the gel showed fragments of expected sizes then the <del class="diffchange diffchange-inline">biobricks </del>transformed into E. coli by Rachel will be used in the ligation of reporters to the hybrid promoter and to carry out quantitative experiments.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Russell carried out a restriction digest of each isolated plasmid, of which we hoped that they hopefully contained the RFP, eCFP or AraC BioBricks. He performed single digests with just EcoRI to linerarise the plasmid and double digests with EcoRI + PstI to cut out the insert. These digested plasmids will then be run on an agarose gel in order to validate the DNA, due to the unusual results previously. If the gel showed fragments of expected sizes then the <ins class="diffchange diffchange-inline">BioBricks </ins>transformed into E. coli by Rachel will be used in the ligation of reporters to the hybrid promoter and to carry out quantitative experiments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A plan was made to characterise PyeaR + GFP (BBa_K1001) and discussed between Lukas, Rebecca and Joy. We plan to grow up PyeaR + GFP in cultures, reading the absorbance at 660nm of the samples regularly (hourly). The inoculation will be made directly from the plate. The pilot study will last for 6 hours and the full study, if the pilot study goes according to plan, will be 12 hours. To generate a comparison of PyeaR + GFP containing cells against non transformed cells, Bioline competent cells were plated and grown for next day use. The final studies and results can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments#A_study_of_growth_of_PyeaR_in_different_concentrations_of_potassium_nitrate here].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A plan was made to characterise PyeaR + GFP (BBa_K1001) and discussed between Lukas, Rebecca and Joy. We plan to grow up PyeaR + GFP in cultures, reading the absorbance at 660nm of the samples regularly (hourly). The inoculation will be made directly from the plate. The pilot study will last for 6 hours and the full study, if the pilot study goes according to plan, will be 12 hours. To generate a comparison of PyeaR + GFP containing cells against non transformed cells, Bioline competent cells were plated and grown for next day use. The final studies and results can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments#A_study_of_growth_of_PyeaR_in_different_concentrations_of_potassium_nitrate here].</div></td></tr>
</table>Khadijaouadihttp://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=195290&oldid=prevLukasHarnisch at 12:33, 26 September 20122012-09-26T12:33:28Z<p></p>
<a href="http://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=195290&oldid=158668">Show changes</a>LukasHarnischhttp://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=158668&oldid=prevLukasHarnisch: /* Labs */2012-09-24T16:28:26Z<p><span class="autocomment">Labs</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The potential BioBricks were then transformed into alpha cells and grown overnight. The protocol used followed that on the Lab Protocol page.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The potential BioBricks were then transformed into alpha cells and grown overnight. The protocol used followed that on the Lab Protocol page.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The pilot study to measure the growth rate of Pyear cells was carried out for the planned 6 hours. From a plate a colony was selected and inoculated into 15ml of LB media in a sterile tube. Into this potassium nitrate was added at 0mm, 5mM and 10mM concentration. ''E.coli'' cells were inoculated into the same amount of media without the addition of potassium nitrate. Every hour an absorbance reading was taken and a serial dilution was made. <del class="diffchange diffchange-inline">TO </del>generate a calibration curve, as we did not know the number of viable cells in relation to the absorbance at 660 nm, was generated plated and measuring the absorbance of various dilutions of the original cell culture.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The pilot study to measure the growth rate of Pyear cells was carried out for the planned 6 hours. From a plate a colony was selected and inoculated into 15ml of LB media in a sterile tube. Into this potassium nitrate was added at 0mm, 5mM and 10mM concentration. ''E.coli'' cells were inoculated into the same amount of media without the addition of potassium nitrate. Every hour an absorbance reading was taken and a serial dilution was made. <ins class="diffchange diffchange-inline">To </ins>generate a calibration curve, as we did not know the number of viable cells in relation to the absorbance at 660 nm, was generated plated and measuring the absorbance of various dilutions of the original cell culture.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Day 5 (10/08/12)==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Day 5 (10/08/12)==</div></td></tr>
</table>LukasHarnischhttp://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=158390&oldid=prevLukasHarnisch: /* Labs */2012-09-24T16:05:25Z<p><span class="autocomment">Labs</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[File:Glow_2.png | thumb | ''An example of a successful transformation with the RFP BioBrick'']]</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Rachel checked the transformed bacteria and found that some samples had grown but others hadn't quite as much; they were left to incubate for a few more hours to grow before inoculation.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Rachel checked the transformed bacteria and found that some samples had grown but others hadn't quite as much; they were left to incubate for a few more hours to grow before inoculation.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The restriction digests (see Friday, Week 4) of the repeated PCR of B-M/M-B was perfromed. Again, it was run on a gel. This time, it showed that the PCR was successful in amplifying the hybrid promoters! </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The restriction digests (see Friday, Week 4) of the repeated PCR of B-M/M-B was perfromed. Again, it was run on a gel. This time, it showed that the PCR was successful in amplifying the hybrid promoters! </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Following validation of the PCR products, we ligated M-B and B-M promoters into pSB1C3. We initially nanodropped each of the samples and used the quantitate data to set up a ligation at a 3:1 ratio between insert to pSB1C3. Positive and negative controls were added to ensure quality control if something did go wrong so that we could determine what caused the error. As s negative control, pSB1C3 would be transformed into alpha gold select cells from Bioline; as a positive control, we ligated PyeaR-GFP into pSB1C3 and transformed the plasmid into alpha cells. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Following validation of the PCR products, we ligated M-B and B-M promoters into pSB1C3. We initially nanodropped each of the samples and used the quantitate data to set up a ligation at a 3:1 ratio between insert to pSB1C3. Positive and negative controls were added to ensure quality control if something did go wrong so that we could determine what caused the error. As s negative control, pSB1C3 would be transformed into alpha gold select cells from Bioline; as a positive control, we ligated PyeaR-GFP into pSB1C3 and transformed the plasmid into alpha cells.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Day 4 (09/08/12)==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Day 4 (09/08/12)==</div></td></tr>
</table>LukasHarnischhttp://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=158361&oldid=prevLukasHarnisch: /* Labs */2012-09-24T16:02:43Z<p><span class="autocomment">Labs</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:Russel+Rachel_gel.png | thumb | '' '''Figure 1.''' Gel electrophoresis of <del class="diffchange diffchange-inline">mini preps </del>of various fluorescent BioBricks (RFP, eCFP and AraC promoter)'']]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:Russel+Rachel_gel.png | thumb | '' '''Figure 1.''' Gel electrophoresis of <ins class="diffchange diffchange-inline">plasmid isolations </ins>of various fluorescent BioBricks (RFP, eCFP and AraC promoter)'']]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Russell and Rachel ran their isolated plasmids containing RFP, eCFP and AraC promoter BioBricks on an agarose gel and found that the bands given were not in-keeping with the size they expected them to be. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not, resulting in the abnormal results and it was concluded that further experiments were needed.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Russell and Rachel ran their isolated plasmids containing RFP, eCFP and AraC promoter BioBricks on an agarose gel and found that the bands given were not in-keeping with the size they expected them to be. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not, resulting in the abnormal results and it was concluded that further experiments were needed <ins class="diffchange diffchange-inline">(''Figure 1.'')</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A gel of the digested PCR products was rerun following the previous Friday's results. Again the gel was unclear so we decided to repeat the PCR restriction digest.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A gel of the digested PCR products was rerun following the previous Friday's results. Again the gel was unclear so we decided to repeat the PCR restriction digest.</div></td></tr>
</table>LukasHarnischhttp://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=158340&oldid=prevLukasHarnisch: /* Labs */2012-09-24T16:01:34Z<p><span class="autocomment">Labs</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[File:Russel+Rachel_gel.png | thumb | '' '''Figure 1.''' Gel electrophoresis of mini preps of various fluorescent BioBricks (RFP, eCFP and AraC promoter)'']]</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Russell and Rachel ran their isolated plasmids containing RFP, eCFP and AraC promoter BioBricks on an agarose gel and found that the bands given were not in-keeping with the size they expected them to be. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not, resulting in the abnormal results and it was concluded that further experiments were needed.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Russell and Rachel ran their isolated plasmids containing RFP, eCFP and AraC promoter BioBricks on an agarose gel and found that the bands given were not in-keeping with the size they expected them to be. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not, resulting in the abnormal results and it was concluded that further experiments were needed.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A gel of the digested PCR products was rerun following the previous Friday's results. Again the gel was unclear so we decided to repeat the PCR restriction digest.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A gel of the digested PCR products was rerun following the previous Friday's results. Again the gel was unclear so we decided to repeat the PCR restriction digest.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The PyeaR + GFP BioBrick in pSB1C3, that had been miniprepped last week, was nanodropped. Following Friday, when the restriction digest of the plasmid with EcoRI and PstI was unsuccessful, we repeated the digest to remove the insert from the backbone. Joy used the nanodropped Pyear + GFP sample. Unlike Friday, the digest was successful. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The PyeaR + GFP BioBrick in pSB1C3, that had been miniprepped last week, was nanodropped. Following Friday, when the restriction digest of the plasmid with EcoRI and PstI was unsuccessful, we repeated the digest to remove the insert from the backbone. Joy used the nanodropped Pyear + GFP sample. Unlike Friday, the digest was successful.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Day 2 (07/08/12)==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Day 2 (07/08/12)==</div></td></tr>
</table>LukasHarnischhttp://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=155934&oldid=prevLukasHarnisch at 12:30, 24 September 20122012-09-24T12:30:43Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">. </del>Rachel and Russell looked at the transformed bacteria and found that the plates had been <del class="diffchange diffchange-inline">infected with something other than the bacteria we were looking for</del>. We had some of the transformant left over so decided to plate again, however the plates that were made were dry/thin. We decided to leave it and <del class="diffchange diffchange-inline">make </del>new plates the next day.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Rachel and Russell looked at the transformed bacteria and found that the plates had been <ins class="diffchange diffchange-inline">contaminated</ins>. We had some of the transformant left over so decided to plate again, however the plates that were made were dry/thin. We decided to leave it and <ins class="diffchange diffchange-inline">prepare </ins>new plates the next day.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">. </del>To test whether the ligation had worked, a restriction digest was run. We digested the samples with BamH1 to linearise. If the ligation was successful, the inserts <del class="diffchange diffchange-inline">would </del>be in pSB1C3 and hence be 2321bp. If unlinearised, <del class="diffchange diffchange-inline">they </del>should travel further due to supercoiling. When linearised they should match this length. Running the products on the restriction digest on a gel showed that we did indeed succeed in ligating B-M and M-B into the iGEM backbone!!! </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To test whether the ligation had worked, a restriction digest was run. We digested the samples with BamH1 to linearise. If the ligation was successful, <ins class="diffchange diffchange-inline">we were expecting </ins>the inserts <ins class="diffchange diffchange-inline">to </ins>be in pSB1C3 and hence be 2321bp <ins class="diffchange diffchange-inline">in size</ins>. If unlinearised, <ins class="diffchange diffchange-inline">the sample </ins>should travel further due to supercoiling. When linearised they should match this length. Running the products on the restriction digest on a gel showed that we did indeed succeed in ligating B-M and M-B into the iGEM backbone!!! </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">. </del>The potential BioBricks were transformed into alpha cells and grown overnight. The protocol used followed that on the Lab Protocol page.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The potential BioBricks were <ins class="diffchange diffchange-inline">then </ins>transformed into alpha cells and grown overnight. The protocol used followed that on the Lab Protocol page.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">. </del>The pilot study was carried out for the planned 6 hours. From <del class="diffchange diffchange-inline">the </del>plate a colony was selected and inoculated into 15ml of LB media in a sterile tube. Into this potassium nitrate was added at 0mm, 5mM and 10mM concentration. ''E.coli'' cells were inoculated into the same amount of media without the addition of potassium nitrate. Every hour an absorbance reading was taken and a serial dilution was made. <del class="diffchange diffchange-inline">Of the dilutions </del>as we <del class="diffchange diffchange-inline">do </del>not know the number of cells to <del class="diffchange diffchange-inline">be expected 3 of </del>the <del class="diffchange diffchange-inline">dilutions were plated (10^-3</del>, <del class="diffchange diffchange-inline">4 </del>and <del class="diffchange diffchange-inline">-5)</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The pilot study <ins class="diffchange diffchange-inline">to measure the growth rate of Pyear cells </ins>was carried out for the planned 6 hours. From <ins class="diffchange diffchange-inline">a </ins>plate a colony was selected and inoculated into 15ml of LB media in a sterile tube. Into this potassium nitrate was added at 0mm, 5mM and 10mM concentration. ''E.coli'' cells were inoculated into the same amount of media without the addition of potassium nitrate. Every hour an absorbance reading was taken and a serial dilution was made. <ins class="diffchange diffchange-inline">TO generate a calibration curve, </ins>as we <ins class="diffchange diffchange-inline">did </ins>not know the number of <ins class="diffchange diffchange-inline">viable </ins>cells <ins class="diffchange diffchange-inline">in relation </ins>to the <ins class="diffchange diffchange-inline">absorbance at 660 nm</ins>, <ins class="diffchange diffchange-inline">was generated plated </ins>and <ins class="diffchange diffchange-inline">measuring the absorbance of various dilutions of the original cell culture</ins>.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Day 5 (10/08/12)==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Day 5 (10/08/12)==</div></td></tr>
</table>LukasHarnischhttp://2012.igem.org/wiki/index.php?title=Team:NRP-UEA-Norwich/Week5&diff=149188&oldid=prevLukasHarnisch at 17:07, 23 September 20122012-09-23T17:07:53Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Labs===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>No growth with the <del class="diffchange diffchange-inline">Biobrick </del>B-M/M-B <del class="diffchange diffchange-inline">- Transformed bacteria, probable </del>issue with the agar as it looked dry<del class="diffchange diffchange-inline">/</del>thin. We still had some bacteria and DNA <del class="diffchange diffchange-inline">for a transformation so re-made </del>the <del class="diffchange diffchange-inline">plates </del>and <del class="diffchange diffchange-inline">decided </del>to <del class="diffchange diffchange-inline">try again </del>with <del class="diffchange diffchange-inline">the transformations and plating in order to see if they grow on </del>new plates.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>No growth <ins class="diffchange diffchange-inline">was observed of the cells transformed </ins>with the <ins class="diffchange diffchange-inline">ligation product of </ins>B-M/M-B <ins class="diffchange diffchange-inline">(PCR) in pSB1C3. We suspected an </ins>issue with the agar as it looked dry <ins class="diffchange diffchange-inline">and </ins>thin. We still had some bacteria and DNA <ins class="diffchange diffchange-inline">left from </ins>the <ins class="diffchange diffchange-inline">previous transformation </ins>and <ins class="diffchange diffchange-inline">were able </ins>to <ins class="diffchange diffchange-inline">re-transform </ins>with new<ins class="diffchange diffchange-inline">, fresh agar </ins>plates <ins class="diffchange diffchange-inline">to double check of the transformation was successful</ins>. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">. </del>Russell and Rachel plated their transformed bacteria and <del class="diffchange diffchange-inline">left to grow </del>over night.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Russell and Rachel plated their transformed bacteria and <ins class="diffchange diffchange-inline">incubated them </ins>over night.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">. </del>The plates from the previous day's pilot study was viewed. It was found that initially, there was an insufficient number of cells in the culture to plate and hence within the first 3 hours, there were little to no cells on the plates. With progression of time there were more colonies.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The plates from the previous day's pilot study <ins class="diffchange diffchange-inline">of PyeaR + GFP </ins>was viewed. It was found that initially, there was an insufficient number of cells in the culture to plate and hence within the first 3 hours, there were little to no cells on the plates. With progression of time there were more colonies.</div></td></tr>
</table>LukasHarnisch