http://2008.igem.org/wiki/index.php?title=Team:UCSF/Leeza_Sergeeva_Notebook&feed=atom&action=history
Team:UCSF/Leeza Sergeeva Notebook - Revision history
2024-03-29T08:06:49Z
Revision history for this page on the wiki
MediaWiki 1.16.5
http://2008.igem.org/wiki/index.php?title=Team:UCSF/Leeza_Sergeeva_Notebook&diff=60730&oldid=prev
Ahorwitz at 23:58, 22 October 2008
2008-10-22T23:58:36Z
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 23:58, 22 October 2008</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><!--- The Mission, Experiments ---></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><!--- The Mission, Experiments ---></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{| style="color:#<ins class="diffchange diffchange-inline">333333</ins>;background-color:#<ins class="diffchange diffchange-inline">cccccc</ins>;" cellpadding="3" cellspacing="<ins class="diffchange diffchange-inline">3</ins>" border="<ins class="diffchange diffchange-inline">0</ins>" bordercolor="#<ins class="diffchange diffchange-inline">231f26</ins>" width="<ins class="diffchange diffchange-inline">99</ins>%" align="center" </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{| style="color:#<del class="diffchange diffchange-inline">1b2c8a</del>;background-color:#<del class="diffchange diffchange-inline">0c6</del>;" cellpadding="3" cellspacing="<del class="diffchange diffchange-inline">1</del>" border="<del class="diffchange diffchange-inline">1</del>" bordercolor="#<del class="diffchange diffchange-inline">fff</del>" width="<del class="diffchange diffchange-inline">62</del>%" align="center"</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: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF|Home]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF|Home]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF/Team|The Team]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF/Team|The Team]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF/Modeling|Modeling]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF/Modeling|Modeling]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF/<del class="diffchange diffchange-inline">Notebook</del>|Notebook]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>!align="center"|[[Team:UCSF/<ins class="diffchange diffchange-inline">Human Practices</ins>|<ins class="diffchange diffchange-inline">Human Practices]]</ins></div></td></tr>
<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">!align="center"|[[Team:UCSF/</ins>Notebook<ins class="diffchange diffchange-inline">|Notebooks</ins>]]</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><del style="color: red; font-weight: bold; text-decoration: none;">(''Or you can choose different headings. But you must have a team page, a project page, and a notebook page.'')</del></div></td><td colspan="2"> </td></tr>
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Ahorwitz
http://2008.igem.org/wiki/index.php?title=Team:UCSF/Leeza_Sergeeva_Notebook&diff=60684&oldid=prev
Raquel Gomes: New page: <html xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns="http://www.w3.org/TR/REC-html40"> <head> <meta http-equiv=Content-Type conte...
2008-10-22T22:31:34Z
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<p class=MsoNormal><span class=SpellE><b style='mso-bidi-font-weight:normal'><u><span<br />
style='font-size:16.0pt;font-family:"Segoe Script"'>Leeza</span></u></b></span><b<br />
style='mso-bidi-font-weight:normal'><u><span style='font-size:16.0pt;<br />
font-family:"Segoe Script"'> <span class=SpellE>Sergeeva</span><o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span<br />
style='font-size:16.0pt;font-family:"Segoe Script"'>Notebook 6/18/08 – 10/24/08<o:p></o:p></span></u></b></p><br />
<br />
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<br />
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span<br />
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<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u>Objective</u></b>:<br />
Disrupt silencing to activate a gene.<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Remodel chromatin so that heterochromatin can be uncoiled<br />
and opened into euchromatin so they can be activated and freely available.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal>We’re <i style='mso-bidi-font-style:normal'>turning on</i><br />
the already silenced genes to produce the desired response (shift the activated<br />
genes towards silencing) by opening heterochromatin and acetylating them to<br />
become euchromatin and open.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>Rationale</b> - In<br />
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gene silencing).<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><u>Milestone</u>: Create multiple fusions of DNA binding<br />
domains and activations domains.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><span class=GramE><u>More specifically…</u></span><u><o:p></o:p></u></p><br />
<br />
<p class=MsoNormal>We are creating our anti-silencing circuits using various<br />
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<br />
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mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'><br />
<p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;height:18.6pt'><br />
<td width=184 valign=top style='width:138.3pt;border:solid windowtext 1.0pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt;<br />
padding:0in 5.4pt 0in 5.4pt;height:18.6pt'><br />
<p class=MsoNormal align=center style='text-align:center'>VP64<o:p></o:p></p><br />
</td><br />
<td width=164 valign=top style='width:122.95pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'><br />
<p class=MsoNormal align=center style='text-align:center'><o:p>&nbsp;</o:p></p><br />
</td><br />
<td width=164 valign=top style='width:122.95pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'><br />
<p class=MsoNormal align=center style='text-align:center'><o:p>&nbsp;</o:p></p><br />
</td><br />
<td width=164 valign=top style='width:123.0pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'><br />
<p class=MsoNormal align=center style='text-align:center'><o:p>&nbsp;</o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;mso-yfti-lastrow:yes;height:18.6pt'><br />
<td width=184 valign=top style='width:138.3pt;border:solid windowtext 1.0pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt;<br />
padding:0in 5.4pt 0in 5.4pt;height:18.6pt'><br />
<p class=MsoNormal align=center style='text-align:center'><span class=SpellE>mCherry</span><o:p></o:p></p><br />
</td><br />
<td width=164 valign=top style='width:122.95pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'><br />
<p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p></p><br />
</td><br />
<td width=164 valign=top style='width:122.95pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'><br />
<p class=MsoNormal align=center style='text-align:center'><o:p>&nbsp;</o:p></p><br />
</td><br />
<td width=164 valign=top style='width:123.0pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'><br />
<p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>(+ - indicates what I’ve finally got)<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><u>The function of each is such</u>:<o:p></o:p></p><br />
<br />
<p class=MsoNormal>1. We’re using various<span lang=ES-VE style='mso-ansi-language:<br />
ES-VE'> LexA</span> domains to compete for the<span lang=EN-GB<br />
style='mso-ansi-language:EN-GB'> <span class=SpellE>LexA</span></span> binding<br />
site. This won’t necessarily open heterochromatin, but it will prevent DNA from<br />
becoming heterochromatin because it will block and HDAC from binding to the <span<br />
class=SpellE>LexA</span> binding site and therefore inhibit the DNA from<br />
becoming silenced.<o:p></o:p></p><br />
<br />
<p class=MsoNormal>2. We’re using <span class=SpellE>HATs</span> such as Sas2<br />
and Esa1 because they will cause an oozing model where we end up acetylating<br />
the heterochromatin and “open” it into euchromatin.<o:p></o:p></p><br />
<br />
<p class=MsoNormal>3. We’re using VP64 - as a reader to attract <span<br />
class=SpellE>HATs</span> (writers) to they will <span class=SpellE>acetylate</span><br />
for us and open the heterochromatin for us indirectly.<o:p></o:p></p><br />
<br />
<p class=MsoNormal>4. We’re using <span class=SpellE>mCherry</span> just as a<br />
reporter of expression level so we can run a <span class=SpellE>FACs</span> on<br />
it.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span<br />
style='font-size:18.0pt;font-family:"Segoe Script"'>Actual work done:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
1-2:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Sequencing VP64<o:p></o:p></p><br />
<br />
<p class=MsoNormal>PCR VP64 <o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
3:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>TOPO clone VP64<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Digest PCR product of VP64 with <span class=SpellE>EcoRI</span><br />
- run gel - VP64 failed<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Bacterial transformation of pFig1<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Miniprep</span> more of p315CYC<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>AarI</span> digestion of p315CYC<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
4:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Run gel to check if <span class=SpellE>AarI</span> digestion<br />
of p315CYC worked (<u>success</u>)<o:p></o:p></p><br />
<br />
<p class=MsoNormal>TOPO clone <span class=SpellE>mCherry</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Miniprep</span> <span class=SpellE>mCherry</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Sequence <span class=SpellE>mCherry</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
5:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal><span class=SpellE>EcoRI</span> Diagnostic Digest of <span<br />
class=SpellE>mCherry</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>AarI</span> digestion of <span<br />
class=SpellE>LexA</span> DBD, <span class=SpellE>LexA</span> FL, <span<br />
class=SpellE>LexA</span> mCherry<span class=GramE>,p315ADH</span>, p305GAL and<br />
VP16<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Run gel on <span class=SpellE>AarI</span> digestion<br />
(success)<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Gel purify <span class=SpellE>LexA</span> DBD, <span<br />
class=SpellE>LexA</span> FL, <span class=SpellE>LexA</span> <span class=SpellE>mCherry</span>,<br />
p315ADH, p305GAL and VP16<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Ligations</span> - failed <o:p></o:p></p><br />
<br />
<p class=MsoNormal><span style='mso-tab-count:1'> </span><span<br />
class=SpellE>Cyc-LexA</span> DBD; <span class=SpellE>Cyc-LexA</span> FL; <span<br />
class=SpellE>Cyc-mCherry</span>; <span class=SpellE>Cyc</span>–VP16<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span style='mso-tab-count:1'> </span>Gal-<span<br />
class=SpellE>LexA</span> DBD; Gal-<span class=SpellE>LexA</span> FL; Gal-<span<br />
class=SpellE>mCherry</span>; Gal-VP16<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE><span class=GramE>AarI</span></span><span<br />
class=GramE> digestion of <span class=SpellE>LexA</span> DBD, VP16, <span<br />
class=SpellE>LexA</span> <span class=SpellE>mCherry</span>, Sas2, <span<br />
class=SpellE>pGal</span>, <span class=SpellE>pAdh</span>, <span class=SpellE>pCyc</span>.</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Rung gel - digestion failed<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Ligations</span> - <u>success</u><o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-LexADBD-VP16<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
FL-VP16<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
mCherry-VP16 <o:p></o:p></span></p><br />
<br />
<p class=MsoNormal>Bacterial Transformation of successful <span class=SpellE>ligations</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.5in'><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
6:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal><span class=SpellE><span class=GramE>AarI</span></span><span<br />
class=GramE> digestion of VP16, Esa1, <span class=SpellE>pGal</span>, <span<br />
class=SpellE>pCyc</span>, Sas2, <span class=SpellE>LexA</span> <span<br />
class=SpellE>mCherry</span>.</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Run Gel - VP16 (failed)<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Colony PCR of <span class=SpellE>ligations</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-LexADBD-VP16<span<br />
style='mso-tab-count:2'> </span><u>Success</u><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
FL-VP16<span style='mso-tab-count:2'> </span><u>Success</u><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
mCherry-VP16 <span style='mso-tab-count:1'> </span><u>Success</u><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal>Yeast transformation of successful <span class=SpellE>ligations</span><br />
into silenced strains:<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span style='mso-tab-count:1'> </span>-<br />
Adh-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span style='mso-tab-count:1'> </span>-<br />
Cyc-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Ligations</span> - <u>success</u><o:p></o:p></p><br />
<br />
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=235<br />
style='width:176.6pt;margin-left:4.75pt;border-collapse:collapse;mso-padding-alt:<br />
0in 5.4pt 0in 5.4pt'><br />
<tr style='mso-yfti-irow:0;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Gal1-LexAmCherry-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Gal1-LexAmCherry-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Gal1-LexA PEG 202 (FL)-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Gal1-LexA PEG 202 (FL)-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Gal1-LexA DBD-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Gal1-LexA DBD-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Adh1-LexAmCherry-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Adh1-LexAmCherry-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:8;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Adh1-LexA PEG 202 (FL)-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:9;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Adh1-LexA PEG 202 (FL)-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:10;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Adh1-LexA DBD-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:11;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Adh1-LexA DBD-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:12;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Cyc1-LexAmCherry-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:13;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Cyc1-LexAmCherry-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:14;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Cyc1-LexA PEG 202 (FL)-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:15;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Cyc1-LexA PEG 202 (FL)-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:16;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Cyc1-LexA DBD-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:17;mso-yfti-lastrow:yes;height:12.3pt'><br />
<td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.3pt'><br />
<p class=MsoNormal>Cyc1-LexA DBD-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
7:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Cut p305Gal with <span class=SpellE>XcmI</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Bacterial Transformation of <span class=SpellE>ligations</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Colonies PCR- <span class=GramE>run</span> gel - unclear<br />
results<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Ligations</span> – <u>success</u> - Bacterial<br />
Transformations - <u>success</u><o:p></o:p></p><br />
<br />
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=220<br />
style='width:165.0pt;margin-left:4.75pt;border-collapse:collapse;mso-padding-alt:<br />
0in 5.4pt 0in 5.4pt'><br />
<tr style='mso-yfti-irow:0;height:12.0pt'><br />
<td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Fig1-LexAmCherry-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1;height:12.0pt'><br />
<td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Fig1-LexAmCherry-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2;height:12.0pt'><br />
<td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Fig1-LexA PEG 202 (FL)-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3;height:12.0pt'><br />
<td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Fig1-LexA PEG 202 (FL)-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;height:12.0pt'><br />
<td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Fig1-LexA DBD-Esa1<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;mso-yfti-lastrow:yes;height:12.0pt'><br />
<td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Fig1-LexA DBD-Sas2<o:p></o:p></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal><span class=SpellE>Miniprep</span> colonies of Gal, <span<br />
class=SpellE><span class=GramE>Adh</span></span> and <span class=SpellE>Cyc</span><br />
<span class=SpellE>ligations</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Diagnostic digest of Gal, <span class=SpellE><span<br />
class=GramE>Adh</span></span> and <span class=SpellE>Cyc</span> <span<br />
class=SpellE>ligations</span> with <span class=SpellE>PspOMI</span> &amp; <span<br />
class=SpellE>SacI</span> - <u>success</u><o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Miniprep</span> Fig <span class=SpellE>ligations</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Diagnostic digest of Fig <span class=SpellE>ligations</span><br />
with <span class=SpellE>XhoI</span> &amp; <span class=SpellE>NotI</span> - <u>success</u><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Repeat of diagnostic digest of Gal, <span class=SpellE><span<br />
class=GramE>Adh</span></span> and <span class=SpellE>Cyc</span> <span<br />
class=SpellE>ligations</span> with <span class=SpellE>PspOMI</span> &amp; <span<br />
class=SpellE>SacI</span> - <u>success</u><o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>AarI</span> digestions of <span<br />
class=SpellE>LexA</span> FL, ADH, VP16, <span class=SpellE>mCherry</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=GramE>Run</span> Gel - <span class=SpellE>LexA</span><br />
FL and ADH failed<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
8:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Yeast transformation of Gal <span class=SpellE>ligations</span><br />
into silenced strains:<o:p></o:p></p><br />
<br />
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=248<br />
style='width:186.0pt;margin-left:4.75pt;border-collapse:collapse;mso-padding-alt:<br />
0in 5.4pt 0in 5.4pt'><br />
<tr style='mso-yfti-irow:0;height:12.0pt'><br />
<td width=248 nowrap valign=bottom style='width:186.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Gal1-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1;height:12.0pt'><br />
<td width=248 nowrap valign=bottom style='width:186.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Adh1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2;height:12.0pt'><br />
<td width=248 nowrap valign=bottom style='width:186.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3;mso-yfti-lastrow:yes;height:12.0pt'><br />
<td width=248 nowrap valign=bottom style='width:186.0pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.0pt'><br />
<p class=MsoNormal>Cyc1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal>Diagnostic digest of Gal <span class=SpellE>Ligations</span><br />
with <span class=SpellE>XhoI</span> &amp; <span class=SpellE>NotI</span> - <u>success</u><o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Restrick</span> Gal yeasts on <span<br />
class=SpellE>SRaf</span> + 2% Gal and SD-<span class=SpellE>Leu</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
9:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Prepare Gal yeasts for <span class=SpellE>FACs</span> in SD<br />
+ 2% Gal and SD-<span class=SpellE>Leu</span> medium.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=GramE><u>Successful <span class=SpellE>FACs</span><br />
results</u> that shows</span> <span class=SpellE>unsilencing</span> of silenced<br />
gene:<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
FL-<span class=SpellE>EsaI</span> on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
FL-Sas2 on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
MC-<span class=SpellE>EsaI</span> on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
FL-<span class=SpellE>EsaI</span> on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
MC-<span class=SpellE>EsaI</span> on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal>Prepare successful <span class=SpellE>FACs</span> results<br />
for repeat <span class=SpellE>FACs</span> experiment in –gal and +gal medium. (<span<br />
class=SpellE>SRaf</span> +2% gal and <span class=SpellE>SDComp</span>)<o:p></o:p></p><br />
<br />
<p class=MsoNormal><u>Successful <span class=SpellE>FACs</span> results</u><br />
that show strong <span class=SpellE>unsilencing</span>:<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
MC-<span class=SpellE>EsaI</span> on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
FL-Sas2 on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal>Set up <span class=SpellE>FACs</span> experiment <span<br />
class=SpellE>unsilencing</span> strain on silencing strain <o:p></o:p></p><br />
<br />
<p class=MsoNormal>Adh-LexA-Sir2/Cyc-5’-GFP:<o:p></o:p></p><br />
<br />
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=235<br />
style='width:176.15pt;border-collapse:collapse;mso-padding-alt:0in 5.4pt 0in 5.4pt'><br />
<tr style='mso-yfti-irow:0;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexAmCherry-Esa1<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexAmCherry-Sas2<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Cyc1-LexA PEG 202<br />
(FL)-Esa1<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Cyc1-LexA PEG 202<br />
(FL)-Sas2<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexA<br />
DBD-Esa1<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexA<br />
DBD-Sas2<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Fig1-LexAmCherry-Esa1<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Fig1-LexAmCherry-Sas2<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:8;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Fig1-LexA PEG 202<br />
(FL)-Esa1<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:9;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Fig1-LexA PEG 202<br />
(FL)-Sas2<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:10;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexA PEG 202<br />
(FL)-Esa1<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:11;mso-yfti-lastrow:yes;height:12.9pt'><br />
<td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt;<br />
height:12.9pt'><br />
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexA PEG 202<br />
(FL)-Sas2<o:p></o:p></span></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p class=MsoNormal><span class=SpellE>FACs</span> results:<o:p></o:p></p><br />
<br />
<p class=MsoNormal>The control strain (silenced strain wasn’t fully silenced)<o:p></o:p></p><br />
<br />
<p class=MsoNormal><u>Discovery</u> - The low concentration of <span<br />
class=SpellE>EsaI</span> induces silencing, but the high <span class=GramE>concentration<br />
<span class=SpellE>unsilence</span></span> the silenced gene.<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Set up cultures for <span class=SpellE><span class=GramE>FACs</span></span><span<br />
class=GramE> :</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:40.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:40.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:40.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
PEG 202 (FL)-Esa1 on silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><span class=SpellE>FACs</span> results - <u>success</u> - <span<br />
class=SpellE>unsilenced</span> silenced genes<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
10:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Transform <i><u><span style='mso-fareast-language:ZH-CN'>Adh1-LexAmCherry-Esa1<br />
(wrong plasmid)</span></u></i><span style='mso-fareast-language:ZH-CN'> and<br />
Cyc1-LexA PEG 202 (FL)-Esa1</span> again into yeast silenced strain<br />
Adh-LexA-Sir2/Cyc-5’-GFP. <span class=GramE>(FAILURE!!!</span> MISTAKE!!! <span<br />
class=GramE>TRANSFORMED WRONG PLASMID!!!)</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal>Prepare Quick <span class=SpellE>Lysates</span> for Western<br />
Blot of:<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Ligations</span><span style='font-family:<br />
Wingdings;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman";<br />
mso-char-type:symbol;mso-symbol-font-family:Wingdings'><span style='mso-char-type:<br />
symbol;mso-symbol-font-family:Wingdings'>à</span></span>Bacterial<br />
transformations - <u>partial success<o:p></o:p></u></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
FL-VP64<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
MC-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
DBD-VP64<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
FL-VP64<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
MC- <span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
DBD-VP64<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span><br />
FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span><br />
FL-VP64<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span><br />
MC- <span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span><br />
DBD-VP64<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Miniprep</span> succeeded <span<br />
class=SpellE>ligations</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal>Cut Gal vectors with <span class=SpellE>XcmI</span> (for<br />
yeast transformation)<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Yeast Transformation of succeeded <span class=SpellE>ligations</span><br />
into new silenced strains (from Alex):<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span style='mso-tab-count:1'> </span>-<br />
Adh-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span style='mso-tab-count:1'> </span>-<br />
Cyc-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Diagnostic Digest of succeeded <span class=SpellE>ligations</span><br />
with <span class=SpellE>PspOMI</span> &amp; <span class=SpellE>SacI</span> - <u>partial<br />
success<o:p></o:p></u></p><br />
<br />
<p class=MsoNormal>Run gel - worked:<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span><br />
MC- <span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span><br />
FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span><br />
MC- <span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal>Set up <span class=SpellE>FACs</span> experiment on <o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:49.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='mso-font-width:0%'><span style='mso-list:Ignore'>-<span<br />
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Adh1-LexA MC-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:49.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1'><![if !supportLists]><span<br />
style='mso-font-width:0%'><span style='mso-list:Ignore'>-<span<br />
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><span class=SpellE>FACs</span> results: New transformations<br />
didn’t work. The control didn’t work.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
11: <o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Set up <span class=SpellE>FACs</span> on new transformation of:<br />
<o:p></o:p></p><br />
<br />
<p class=MsoNormal>- Silenced strain Adh-LexA-Sir2/Cyc-5’-GFP (Medium: SD-<span<br />
class=SpellE>Leu</span> +0.5% <span class=SpellE>Glu</span>)<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
FL-<span class=SpellE>mCherry</span> <o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
MC-Esa1 <o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Cyc</span>- <span<br />
class=SpellE>LexA</span> FL-Esa1<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal>- Silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP (Medium: <span<br />
class=SpellE>SRaf</span> Comp +2% Gal)<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Cyc</span>- <span<br />
class=SpellE>LexA</span> FL-Esa1<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><span class=SpellE>FACs</span> results: Too much background -<br />
unclear results<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>AarI</span> digestion of VP64 - run gel -<br />
couldn’t cut and gel purify VP64<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
12:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Digest Fig-<span class=SpellE>LexA</span> FL-<span<br />
class=SpellE>mCherry</span> &amp; Fig-<span class=SpellE>LexA</span> MC-<span<br />
class=SpellE>mCherry</span> with <span class=SpellE>PspOMI</span> and <span<br />
class=SpellE>SacI</span><o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Ligation</span> into 2m plasmid:<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span><br />
FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span><br />
MC-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal>Set up <span class=SpellE>FACs</span> on:<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
MC-Esa1<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
FL-Esa1<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
DBD-Esa1<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span><br />
FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><span class=SpellE>FACs</span> results: control wasn’t<br />
perfect - <span class=SpellE><span class=GramE>Adh-</span>LexA</span> MC-Esa1<br />
and <span class=SpellE>Adh-LexA</span> FL-<span class=SpellE>mCherry</span><br />
seems to be working.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
13:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Run Western Blot on prepared samples of <o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><span class=GramE>for</span> detecting <span class=SpellE>LexA</span><br />
and <span class=SpellE>Hexokinase</span>. <o:p></o:p></p><br />
<br />
<p class=MsoNormal>Result - <u>partial success</u> - western blot worked - <span<br />
class=SpellE>hexokinase</span> showed up, but the <span class=SpellE>LexA</span><br />
protein wasn’t detected.<o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
14 – 18:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Repeat western blot <span style='font-family:Wingdings;<br />
mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman";<br />
mso-char-type:symbol;mso-symbol-font-family:Wingdings'><span style='mso-char-type:<br />
symbol;mso-symbol-font-family:Wingdings'>à</span></span> gel failed to transfer<br />
to the paper.<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Prepare new Quick <span class=SpellE>Lysates</span> for<br />
Western Blot of:<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language:<br />
ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
19:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Testing the shuttle vectors from AO<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span style='mso-tab-count:1'> </span>Andrew has<br />
done:<o:p></o:p></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l0 level1 lfo2;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>1)<span<br />
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]><span<br />
dir=LTR>Cut AO shuttle vectors with <span class=SpellE>AarI</span>, <span<br />
class=SpellE>XmaI</span> digested, CIP treated, gel purified.<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l0 level1 lfo2;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>2)<span<br />
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]><span<br />
dir=LTR>Cut AB and BD donors with <span class=SpellE>AarI</span>, gel purified.<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l0 level1 lfo2;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>3)<span<br />
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]><span<br />
dir=LTR><span class=SpellE>Ligations</span><o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Plate 1: AB shuttle + AB <span<br />
class=SpellE>LacI</span> - 1107bp<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Plate 2: AB shuttle + <span<br />
class=SpellE>LexA</span> (1-261) - 261bp (Probably FAILED)<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Plate 3: BD shuttle + RPD3cat -<br />
1000bp<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1;<br />
tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span<br />
style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span dir=LTR>Plate 4: BD shuttle + <span<br />
class=SpellE>LexA</span> (1-261) - 261bp<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal>I continued work:<o:p></o:p></p><br />
<br />
<p class=MsoNormal><span class=SpellE>Miniprep</span> Plates 1-4<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Digest vectors with <span class=SpellE>EcoRI</span> &amp; <span<br />
class=SpellE>SpeI</span> - Run gel - digestion failed<o:p></o:p></p><br />
<br />
<p class=MsoNormal>Repeat digestion with <span class=SpellE>EcoRI</span> &amp; <span<br />
class=SpellE>SpeI</span> - Run gel – <u>success<o:p></o:p></u></p><br />
<br />
<p class=MsoNormal><u><o:p><span style='text-decoration:none'>&nbsp;</span></o:p></u></p><br />
<br />
<p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week<br />
20:<o:p></o:p></span></u></b></p><br />
<br />
<p class=MsoNormal>Run the final gel that shows <span class=SpellE>AarI</span><br />
cut vectors and <span class=SpellE>EcoRI</span> &amp; <span class=SpellE>SpeI</span><br />
Shuttle vectors - <u>success</u><o:p></o:p></p><br />
<br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<br />
</div><br />
<br />
</body><br />
<br />
</html><br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:UCSF|Home]]<br />
!align="center"|[[Team:UCSF/Team|The Team]]<br />
!align="center"|[[Team:UCSF/Project|The Project]]<br />
!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:UCSF/Modeling|Modeling]]<br />
!align="center"|[[Team:UCSF/Notebook|Notebook]]<br />
|}<br />
(''Or you can choose different headings. But you must have a team page, a project page, and a notebook page.'')</div>
Raquel Gomes