http://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&feed=atom&action=historyFACS Analysis of mOrange recombinant pRS415 - Revision history2024-03-28T16:55:41ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=194778&oldid=prevKrystal at 20:11, 27 October 20102010-10-27T20:11:37Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the <a href="https://2010.igem.org/FACS_analysis_of_fluorescent_proteins"><i><del class="diffchange diffchange-inline">Flow cytometry</del></i></a></> analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively, (in terms of excitation/emission spectra and filters available) as can be seen from the following data obtained from the BD Fluorescence Spectrum Viewer (Fig.1).</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the <a href="https://2010.igem.org/FACS_analysis_of_fluorescent_proteins"><i><ins class="diffchange diffchange-inline">FACS</ins></i></a></> analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively, (in terms of excitation/emission spectra and filters available) as can be seen from the following data obtained from the BD Fluorescence Spectrum Viewer (Fig.1).</p></div></td></tr>
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</table>Krystalhttp://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=194740&oldid=prevKrystal at 20:10, 27 October 20102010-10-27T20:10:10Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the <del class="diffchange diffchange-inline">FACs </del>analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively, (in terms of excitation/emission spectra and filters available) as can be seen from the following data obtained from the BD Fluorescence Spectrum Viewer (Fig.1).</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the <ins class="diffchange diffchange-inline"><a href="https://2010.igem.org/FACS_analysis_of_fluorescent_proteins"><i>Flow cytometry</i></a></> </ins>analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively, (in terms of excitation/emission spectra and filters available) as can be seen from the following data obtained from the BD Fluorescence Spectrum Viewer (Fig.1).</p></div></td></tr>
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</table>Krystalhttp://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=194703&oldid=prevKrystal at 20:09, 27 October 20102010-10-27T20:09:03Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>From the <ins class="diffchange diffchange-inline"><a href="https://2010.igem.org/FACS_analysis_of_fluorescent_proteins"><i></ins>FACS<ins class="diffchange diffchange-inline"></i></a></> </ins>analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p><br></div></td></tr>
</table>Krystalhttp://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=177957&oldid=prevSlam at 09:41, 27 October 20102010-10-27T09:41:18Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the FACs analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively as can be seen from the following data obtained from the BD <del class="diffchange diffchange-inline">Fluoresence </del>Spectrum Viewer (Fig.1).</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the FACs analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively<ins class="diffchange diffchange-inline">, (in terms of excitation/emission spectra and filters available) </ins>as can be seen from the following data obtained from the BD <ins class="diffchange diffchange-inline">Fluorescence </ins>Spectrum Viewer (Fig.1).</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p><br></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 class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to <del class="diffchange diffchange-inline">autofluorescence </del>from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p><br> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to <ins class="diffchange diffchange-inline">auto-fluorescence </ins>from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p><br> </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>An analysis for GAL1p-[Npep-mOran] sample 6 and 7 using FITC to show that there was no GFP fluorescence was also carried out as shown in Fig.2(ii). However, a small peak was observed for sample 6, which was unexpected. This may have been because the FITC filters overlap the emission spectra of mOrange as shown previously, (BD Spectrum Viewer). Further analysis using the PE-A filter specific for mOrange however show that there is a less well defined and smaller peak for sample 6 than would be expected if there was mOrange fluorescence. This is shown in Fig.3(ii), which suggests that the fluorescence detected is GFP, which was not expected.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>An analysis for GAL1p-[Npep-mOran] sample 6 and 7 using FITC to show that there was no GFP fluorescence was also carried out as shown in Fig.2(ii). However, a small peak was observed for sample 6, which was unexpected. This may have been because the FITC filters overlap the emission spectra of mOrange as shown previously, (BD Spectrum Viewer). Further analysis using the PE-A filter specific for mOrange however show that there is a less well defined and smaller peak for sample 6 than would be expected if there was mOrange fluorescence. This is shown in Fig.3(ii), which suggests that the fluorescence detected is GFP, which was not expected.</p><br></div></td></tr>
</table>Slamhttp://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=158570&oldid=prevPorter at 12:02, 26 October 20102010-10-26T12:02:16Z<p></p>
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</table>Porterhttp://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=158427&oldid=prevPorter at 11:51, 26 October 20102010-10-26T11:51:51Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h3>References</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h3>References<ins class="diffchange diffchange-inline"></h3></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><sup style="font-size:10px">[1]</sup><a href="http://www.bdbiosciences.com/external_files/media/spectrumviewer/index.jsp">Click here to visit the BD Website</a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><sup style="font-size:10px">[1]</sup><a href="http://www.bdbiosciences.com/external_files/media/spectrumviewer/index.jsp">Click here to visit the BD Website</a></p></div></td></tr>
</table>Porterhttp://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=158421&oldid=prevPorter at 11:51, 26 October 20102010-10-26T11:51:18Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusions</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Conclusions</h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. </div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><h3>References</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td></tr>
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</table>Porterhttp://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=158396&oldid=prevPorter at 11:49, 26 October 20102010-10-26T11:49:01Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. </div></td></tr>
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</table>Porterhttp://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=124287&oldid=prevSlam at 12:09, 23 October 20102010-10-23T12:09:24Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p><br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p><ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to autofluorescence from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p><br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to autofluorescence from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p><br> </div></td></tr>
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</table>Slamhttp://2010.igem.org/wiki/index.php?title=FACS_Analysis_of_mOrange_recombinant_pRS415&diff=124285&oldid=prevSlam at 12:08, 23 October 20102010-10-23T12:08:52Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Results</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p><ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to autofluorescence from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to autofluorescence from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p><ins class="diffchange diffchange-inline"><br> </ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>An analysis for GAL1p-[Npep-mOran] sample 6 and 7 using FITC to show that there was no GFP fluorescence was also carried out as shown in Fig.2(ii). However, a small peak was observed for sample 6, which was unexpected. This may have been because the FITC filters overlap the emission spectra of mOrange as shown previously, (BD Spectrum Viewer). Further analysis using the PE-A filter specific for mOrange however show that there is a less well defined and smaller peak for sample 6 than would be expected if there was mOrange fluorescence. This is shown in Fig.3(ii), which suggests that the fluorescence detected is GFP, which was not expected.</p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; 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><p>In a previous experiment, PCR screening was used to establish that the transformed colonies were positive for GAL1p-[Npep-mOran], which makes the chances of a BY4741 transformed with uncut and non-homologously recombined GAL1p-[Npep-GFP] with mOrange insert unlikely.</p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>An analysis for GAL1p-[Npep-mOran] sample 6 and 7 using FITC to show that there was no GFP fluorescence was also carried out as shown in Fig.2(ii). However, a small peak was observed for sample 6, which was unexpected. This may have been because the FITC filters overlap the emission spectra of mOrange as shown previously, (BD Spectrum Viewer). Further analysis using the PE-A filter specific for mOrange however show that there is a less well defined and smaller peak for sample 6 than would be expected if there was mOrange fluorescence. This is shown in Fig.3(ii), which suggests that the fluorescence detected is GFP, which was not expected.</p><ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>In a previous experiment, PCR screening was used to establish that the transformed colonies were positive for GAL1p-[Npep-mOran], which makes the chances of a BY4741 transformed with uncut and non-homologously recombined GAL1p-[Npep-GFP] with mOrange insert unlikely.</p><ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Despite this, the observation of a small peak for both FITC and PE-A with the features as discussed above suggests that there are some yeast cells within the GAL1p-[Npep-mOran] BY4741 samples expressing GFP rather than mOrange. Which implies that the homologous recombination was not 100% efficient.</p> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Despite this, the observation of a small peak for both FITC and PE-A with the features as discussed above suggests that there are some yeast cells within the GAL1p-[Npep-mOran] BY4741 samples expressing GFP rather than mOrange. Which implies that the homologous recombination was not 100% efficient.</p> </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Conversely, 83.4% of the analysed cell population did not show any fluorescence, which suggests that either mOrange was not successfully homologously recombined or that it could not be expressed by BY4741. If uncut GAL1p-[Npep-GFP] had been transformed in place of GAL1p-[Npep-mOran] it is likely that a greater GFP fluorescence would have been detected, which was not the case. The cut GAL1p-[Npep-GFP] does not have any complementary ends that could re-ligate. Therefore it is unlikely that these are transformed and selected.</p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Conversely, 83.4% of the analysed cell population did not show any fluorescence, which suggests that either mOrange was not successfully homologously recombined or that it could not be expressed by BY4741. If uncut GAL1p-[Npep-GFP] had been transformed in place of GAL1p-[Npep-mOran] it is likely that a greater GFP fluorescence would have been detected, which was not the case. The cut GAL1p-[Npep-GFP] does not have any complementary ends that could re-ligate. Therefore it is unlikely that these are transformed and selected.</p><ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Ideally, a positive control for detecting mOrange would allow verification of the specificity of PE-A detection of mOrange and a DNA sequence of transformant of GAL1p-[Npep-mOran] would confirm whether or not the yeast carried the appropriate recombined plasmids.</p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Ideally, a positive control for detecting mOrange would allow verification of the specificity of PE-A detection of mOrange and a DNA sequence of transformant of GAL1p-[Npep-mOran] would confirm whether or not the yeast carried the appropriate recombined plasmids.</p<ins class="diffchange diffchange-inline">><br</ins>> </div></td></tr>
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</table>Slam