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Leeza Sergeeva
From 2008.igem.org
(Difference between revisions)
(New page: LEEZA SERGEEVA'S NOTEBOOK <!--- The Mission, Experiments ---> {| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" ...) |
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| + | <title>Elizaveta Sergeeva</title> | ||
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| + | <p class=MsoNormal><span class=SpellE><b style='mso-bidi-font-weight:normal'><u><span | ||
| + | style='font-size:16.0pt;font-family:"Segoe Script"'>Leeza</span></u></b></span><b | ||
| + | style='mso-bidi-font-weight:normal'><u><span style='font-size:16.0pt; | ||
| + | font-family:"Segoe Script"'> <span class=SpellE>Sergeeva</span><o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span | ||
| + | style='font-size:16.0pt;font-family:"Segoe Script"'>Notebook 6/18/08 – 10/24/08<o:p></o:p></span></u></b></p> | ||
| + | |||
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| + | |||
| + | <p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span | ||
| + | style='font-size:16.0pt;font-family:"Segoe Script"'>Milestones:<o:p></o:p></span></u></b></p> | ||
| + | |||
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| + | |||
| + | <p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u>Objective</u></b>: | ||
| + | Disrupt silencing to activate a gene.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Remodel chromatin so that heterochromatin can be uncoiled | ||
| + | and opened into euchromatin so they can be activated and freely available.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>We’re <i style='mso-bidi-font-style:normal'>turning on</i> | ||
| + | the already silenced genes to produce the desired response (shift the activated | ||
| + | genes towards silencing) by opening heterochromatin and acetylating them to | ||
| + | become euchromatin and open.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b style='mso-bidi-font-weight:normal'>Rationale</b> - In | ||
| + | some cases, we will want differentiation to lead to gene expression (instead of | ||
| + | gene silencing).<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><u>Milestone</u>: Create multiple fusions of DNA binding | ||
| + | domains and activations domains.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=GramE><u>More specifically…</u></span><u><o:p></o:p></u></p> | ||
| + | |||
| + | <p class=MsoNormal>We are creating our anti-silencing circuits using various | ||
| + | DNA Binding Domains and activation domains. (As shown below)<o:p></o:p></p> | ||
| + | |||
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| + | <p class=MsoNormal><o:p> </o:p></p> | ||
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| + | <p class=MsoNormal align=center style='text-align:center'>DNA Binding Domains<o:p></o:p></p> | ||
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| + | <p class=MsoNormal>Activation domains |<br style='mso-special-character:line-break'> | ||
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| + | <p class=MsoNormal><span class=SpellE>LexA</span> PEG 202 (Full Length) NLS<o:p></o:p></p> | ||
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| + | <p class=MsoNormal><span class=SpellE>LexA</span> 1-261 (DBD-short length) | ||
| + | NLS<o:p></o:p></p> | ||
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| + | <p class=MsoNormal><span class=SpellE>LexA</span> 1-261 <span class=SpellE>mCherry</span> | ||
| + | NLS<o:p></o:p></p> | ||
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| + | mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; | ||
| + | mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> | ||
| + | <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p></p> | ||
| + | </td> | ||
| + | <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: | ||
| + | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
| + | mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; | ||
| + | mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> | ||
| + | <p class=MsoNormal align=center style='text-align:center'><o:p> </o:p></p> | ||
| + | </td> | ||
| + | <td width=164 valign=top style='width:123.0pt;border-top:none;border-left: | ||
| + | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
| + | mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; | ||
| + | mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> | ||
| + | <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <p class=MsoNormal>(+ - indicates what I’ve finally got)<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><u>The function of each is such</u>:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>1. We’re using various<span lang=ES-VE style='mso-ansi-language: | ||
| + | ES-VE'> LexA</span> domains to compete for the<span lang=EN-GB | ||
| + | style='mso-ansi-language:EN-GB'> <span class=SpellE>LexA</span></span> binding | ||
| + | site. This won’t necessarily open heterochromatin, but it will prevent DNA from | ||
| + | becoming heterochromatin because it will block and HDAC from binding to the <span | ||
| + | class=SpellE>LexA</span> binding site and therefore inhibit the DNA from | ||
| + | becoming silenced.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>2. We’re using <span class=SpellE>HATs</span> such as Sas2 | ||
| + | and Esa1 because they will cause an oozing model where we end up acetylating | ||
| + | the heterochromatin and “open” it into euchromatin.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>3. We’re using VP64 - as a reader to attract <span | ||
| + | class=SpellE>HATs</span> (writers) to they will <span class=SpellE>acetylate</span> | ||
| + | for us and open the heterochromatin for us indirectly.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>4. We’re using <span class=SpellE>mCherry</span> just as a | ||
| + | reporter of expression level so we can run a <span class=SpellE>FACs</span> on | ||
| + | it.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span | ||
| + | style='font-size:18.0pt;font-family:"Segoe Script"'>Actual work done:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 1-2:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Sequencing VP64<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>PCR VP64 <o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 3:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>TOPO clone VP64<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Digest PCR product of VP64 with <span class=SpellE>EcoRI</span> | ||
| + | - run gel - VP64 failed<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Bacterial transformation of pFig1<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Miniprep</span> more of p315CYC<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>AarI</span> digestion of p315CYC<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 4:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Run gel to check if <span class=SpellE>AarI</span> digestion | ||
| + | of p315CYC worked (<u>success</u>)<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>TOPO clone <span class=SpellE>mCherry</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Miniprep</span> <span class=SpellE>mCherry</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Sequence <span class=SpellE>mCherry</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 5:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>EcoRI</span> Diagnostic Digest of <span | ||
| + | class=SpellE>mCherry</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>AarI</span> digestion of <span | ||
| + | class=SpellE>LexA</span> DBD, <span class=SpellE>LexA</span> FL, <span | ||
| + | class=SpellE>LexA</span> mCherry<span class=GramE>,p315ADH</span>, p305GAL and | ||
| + | VP16<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Run gel on <span class=SpellE>AarI</span> digestion | ||
| + | (success)<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Gel purify <span class=SpellE>LexA</span> DBD, <span | ||
| + | class=SpellE>LexA</span> FL, <span class=SpellE>LexA</span> <span class=SpellE>mCherry</span>, | ||
| + | p315ADH, p305GAL and VP16<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Ligations</span> - failed <o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span style='mso-tab-count:1'> </span><span | ||
| + | class=SpellE>Cyc-LexA</span> DBD; <span class=SpellE>Cyc-LexA</span> FL; <span | ||
| + | class=SpellE>Cyc-mCherry</span>; <span class=SpellE>Cyc</span>–VP16<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span style='mso-tab-count:1'> </span>Gal-<span | ||
| + | class=SpellE>LexA</span> DBD; Gal-<span class=SpellE>LexA</span> FL; Gal-<span | ||
| + | class=SpellE>mCherry</span>; Gal-VP16<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE><span class=GramE>AarI</span></span><span | ||
| + | class=GramE> digestion of <span class=SpellE>LexA</span> DBD, VP16, <span | ||
| + | class=SpellE>LexA</span> <span class=SpellE>mCherry</span>, Sas2, <span | ||
| + | class=SpellE>pGal</span>, <span class=SpellE>pAdh</span>, <span class=SpellE>pCyc</span>.</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Rung gel - digestion failed<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Ligations</span> - <u>success</u><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-LexADBD-VP16<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | FL-VP16<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | mCherry-VP16 <o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal>Bacterial Transformation of successful <span class=SpellE>ligations</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.5in'><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 6:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE><span class=GramE>AarI</span></span><span | ||
| + | class=GramE> digestion of VP16, Esa1, <span class=SpellE>pGal</span>, <span | ||
| + | class=SpellE>pCyc</span>, Sas2, <span class=SpellE>LexA</span> <span | ||
| + | class=SpellE>mCherry</span>.</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Run Gel - VP16 (failed)<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Colony PCR of <span class=SpellE>ligations</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-LexADBD-VP16<span | ||
| + | style='mso-tab-count:2'> </span><u>Success</u><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | FL-VP16<span style='mso-tab-count:2'> </span><u>Success</u><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | mCherry-VP16 <span style='mso-tab-count:1'> </span><u>Success</u><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal>Yeast transformation of successful <span class=SpellE>ligations</span> | ||
| + | into silenced strains:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span style='mso-tab-count:1'> </span>- | ||
| + | Adh-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span style='mso-tab-count:1'> </span>- | ||
| + | Cyc-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Ligations</span> - <u>success</u><o:p></o:p></p> | ||
| + | |||
| + | <table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=235 | ||
| + | style='width:176.6pt;margin-left:4.75pt;border-collapse:collapse;mso-padding-alt: | ||
| + | 0in 5.4pt 0in 5.4pt'> | ||
| + | <tr style='mso-yfti-irow:0;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Gal1-LexAmCherry-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:1;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Gal1-LexAmCherry-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:2;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Gal1-LexA PEG 202 (FL)-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:3;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Gal1-LexA PEG 202 (FL)-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:4;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Gal1-LexA DBD-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:5;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Gal1-LexA DBD-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:6;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Adh1-LexAmCherry-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:7;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Adh1-LexAmCherry-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:8;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Adh1-LexA PEG 202 (FL)-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:9;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Adh1-LexA PEG 202 (FL)-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:10;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Adh1-LexA DBD-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:11;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Adh1-LexA DBD-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:12;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Cyc1-LexAmCherry-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:13;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Cyc1-LexAmCherry-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:14;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Cyc1-LexA PEG 202 (FL)-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:15;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Cyc1-LexA PEG 202 (FL)-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:16;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Cyc1-LexA DBD-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:17;mso-yfti-lastrow:yes;height:12.3pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.6pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.3pt'> | ||
| + | <p class=MsoNormal>Cyc1-LexA DBD-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 7:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Cut p305Gal with <span class=SpellE>XcmI</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Bacterial Transformation of <span class=SpellE>ligations</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Colonies PCR- <span class=GramE>run</span> gel - unclear | ||
| + | results<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Ligations</span> – <u>success</u> - Bacterial | ||
| + | Transformations - <u>success</u><o:p></o:p></p> | ||
| + | |||
| + | <table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=220 | ||
| + | style='width:165.0pt;margin-left:4.75pt;border-collapse:collapse;mso-padding-alt: | ||
| + | 0in 5.4pt 0in 5.4pt'> | ||
| + | <tr style='mso-yfti-irow:0;height:12.0pt'> | ||
| + | <td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Fig1-LexAmCherry-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:1;height:12.0pt'> | ||
| + | <td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Fig1-LexAmCherry-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:2;height:12.0pt'> | ||
| + | <td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Fig1-LexA PEG 202 (FL)-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:3;height:12.0pt'> | ||
| + | <td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Fig1-LexA PEG 202 (FL)-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:4;height:12.0pt'> | ||
| + | <td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Fig1-LexA DBD-Esa1<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:5;mso-yfti-lastrow:yes;height:12.0pt'> | ||
| + | <td width=220 nowrap valign=bottom style='width:165.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Fig1-LexA DBD-Sas2<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Miniprep</span> colonies of Gal, <span | ||
| + | class=SpellE><span class=GramE>Adh</span></span> and <span class=SpellE>Cyc</span> | ||
| + | <span class=SpellE>ligations</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Diagnostic digest of Gal, <span class=SpellE><span | ||
| + | class=GramE>Adh</span></span> and <span class=SpellE>Cyc</span> <span | ||
| + | class=SpellE>ligations</span> with <span class=SpellE>PspOMI</span> & <span | ||
| + | class=SpellE>SacI</span> - <u>success</u><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Miniprep</span> Fig <span class=SpellE>ligations</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Diagnostic digest of Fig <span class=SpellE>ligations</span> | ||
| + | with <span class=SpellE>XhoI</span> & <span class=SpellE>NotI</span> - <u>success</u><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Repeat of diagnostic digest of Gal, <span class=SpellE><span | ||
| + | class=GramE>Adh</span></span> and <span class=SpellE>Cyc</span> <span | ||
| + | class=SpellE>ligations</span> with <span class=SpellE>PspOMI</span> & <span | ||
| + | class=SpellE>SacI</span> - <u>success</u><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>AarI</span> digestions of <span | ||
| + | class=SpellE>LexA</span> FL, ADH, VP16, <span class=SpellE>mCherry</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=GramE>Run</span> Gel - <span class=SpellE>LexA</span> | ||
| + | FL and ADH failed<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 8:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Yeast transformation of Gal <span class=SpellE>ligations</span> | ||
| + | into silenced strains:<o:p></o:p></p> | ||
| + | |||
| + | <table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=248 | ||
| + | style='width:186.0pt;margin-left:4.75pt;border-collapse:collapse;mso-padding-alt: | ||
| + | 0in 5.4pt 0in 5.4pt'> | ||
| + | <tr style='mso-yfti-irow:0;height:12.0pt'> | ||
| + | <td width=248 nowrap valign=bottom style='width:186.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Gal1-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:1;height:12.0pt'> | ||
| + | <td width=248 nowrap valign=bottom style='width:186.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Adh1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:2;height:12.0pt'> | ||
| + | <td width=248 nowrap valign=bottom style='width:186.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:3;mso-yfti-lastrow:yes;height:12.0pt'> | ||
| + | <td width=248 nowrap valign=bottom style='width:186.0pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.0pt'> | ||
| + | <p class=MsoNormal>Cyc1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <p class=MsoNormal>Diagnostic digest of Gal <span class=SpellE>Ligations</span> | ||
| + | with <span class=SpellE>XhoI</span> & <span class=SpellE>NotI</span> - <u>success</u><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Restrick</span> Gal yeasts on <span | ||
| + | class=SpellE>SRaf</span> + 2% Gal and SD-<span class=SpellE>Leu</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 9:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Prepare Gal yeasts for <span class=SpellE>FACs</span> in SD | ||
| + | + 2% Gal and SD-<span class=SpellE>Leu</span> medium.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=GramE><u>Successful <span class=SpellE>FACs</span> | ||
| + | results</u> that shows</span> <span class=SpellE>unsilencing</span> of silenced | ||
| + | gene:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | FL-<span class=SpellE>EsaI</span> on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | FL-Sas2 on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | MC-<span class=SpellE>EsaI</span> on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | FL-<span class=SpellE>EsaI</span> on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | MC-<span class=SpellE>EsaI</span> on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal>Prepare successful <span class=SpellE>FACs</span> results | ||
| + | for repeat <span class=SpellE>FACs</span> experiment in –gal and +gal medium. (<span | ||
| + | class=SpellE>SRaf</span> +2% gal and <span class=SpellE>SDComp</span>)<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><u>Successful <span class=SpellE>FACs</span> results</u> | ||
| + | that show strong <span class=SpellE>unsilencing</span>:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | MC-<span class=SpellE>EsaI</span> on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | FL-Sas2 on Gal10-LexA-Sir2 / <span class=SpellE>Cyc</span>-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal>Set up <span class=SpellE>FACs</span> experiment <span | ||
| + | class=SpellE>unsilencing</span> strain on silencing strain <o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Adh-LexA-Sir2/Cyc-5’-GFP:<o:p></o:p></p> | ||
| + | |||
| + | <table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width=235 | ||
| + | style='width:176.15pt;border-collapse:collapse;mso-padding-alt:0in 5.4pt 0in 5.4pt'> | ||
| + | <tr style='mso-yfti-irow:0;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexAmCherry-Esa1<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:1;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexAmCherry-Sas2<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:2;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Cyc1-LexA PEG 202 | ||
| + | (FL)-Esa1<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:3;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Cyc1-LexA PEG 202 | ||
| + | (FL)-Sas2<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:4;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexA | ||
| + | DBD-Esa1<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:5;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexA | ||
| + | DBD-Sas2<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:6;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Fig1-LexAmCherry-Esa1<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:7;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Fig1-LexAmCherry-Sas2<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:8;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Fig1-LexA PEG 202 | ||
| + | (FL)-Esa1<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:9;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Fig1-LexA PEG 202 | ||
| + | (FL)-Sas2<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:10;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexA PEG 202 | ||
| + | (FL)-Esa1<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:11;mso-yfti-lastrow:yes;height:12.9pt'> | ||
| + | <td width=235 nowrap valign=bottom style='width:176.15pt;padding:0in 5.4pt 0in 5.4pt; | ||
| + | height:12.9pt'> | ||
| + | <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Adh1-LexA PEG 202 | ||
| + | (FL)-Sas2<o:p></o:p></span></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>FACs</span> results:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>The control strain (silenced strain wasn’t fully silenced)<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><u>Discovery</u> - The low concentration of <span | ||
| + | class=SpellE>EsaI</span> induces silencing, but the high <span class=GramE>concentration | ||
| + | <span class=SpellE>unsilence</span></span> the silenced gene.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Set up cultures for <span class=SpellE><span class=GramE>FACs</span></span><span | ||
| + | class=GramE> :</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:40.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:40.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:40.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | PEG 202 (FL)-Esa1 on silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>FACs</span> results - <u>success</u> - <span | ||
| + | class=SpellE>unsilenced</span> silenced genes<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 10:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Transform <i><u><span style='mso-fareast-language:ZH-CN'>Adh1-LexAmCherry-Esa1 | ||
| + | (wrong plasmid)</span></u></i><span style='mso-fareast-language:ZH-CN'> and | ||
| + | Cyc1-LexA PEG 202 (FL)-Esa1</span> again into yeast silenced strain | ||
| + | Adh-LexA-Sir2/Cyc-5’-GFP. <span class=GramE>(FAILURE!!!</span> MISTAKE!!! <span | ||
| + | class=GramE>TRANSFORMED WRONG PLASMID!!!)</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Prepare Quick <span class=SpellE>Lysates</span> for Western | ||
| + | Blot of:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .5in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Ligations</span><span style='font-family: | ||
| + | Wingdings;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; | ||
| + | mso-char-type:symbol;mso-symbol-font-family:Wingdings'><span style='mso-char-type: | ||
| + | symbol;mso-symbol-font-family:Wingdings'>à</span></span>Bacterial | ||
| + | transformations - <u>partial success<o:p></o:p></u></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | FL-VP64<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | MC-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | DBD-VP64<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | FL-VP64<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | MC- <span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | DBD-VP64<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span> | ||
| + | FL-VP64<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span> | ||
| + | MC- <span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span> | ||
| + | DBD-VP64<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Miniprep</span> succeeded <span | ||
| + | class=SpellE>ligations</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Cut Gal vectors with <span class=SpellE>XcmI</span> (for | ||
| + | yeast transformation)<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Yeast Transformation of succeeded <span class=SpellE>ligations</span> | ||
| + | into new silenced strains (from Alex):<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span style='mso-tab-count:1'> </span>- | ||
| + | Adh-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span style='mso-tab-count:1'> </span>- | ||
| + | Cyc-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Diagnostic Digest of succeeded <span class=SpellE>ligations</span> | ||
| + | with <span class=SpellE>PspOMI</span> & <span class=SpellE>SacI</span> - <u>partial | ||
| + | success<o:p></o:p></u></p> | ||
| + | |||
| + | <p class=MsoNormal>Run gel - worked:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Gal-<span class=SpellE>LexA</span> | ||
| + | MC- <span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span> | ||
| + | MC- <span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal>Set up <span class=SpellE>FACs</span> experiment on <o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:49.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1'><![if !supportLists]><span | ||
| + | style='mso-font-width:0%'><span style='mso-list:Ignore'>-<span | ||
| + | style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Adh1-LexA MC-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:49.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1'><![if !supportLists]><span | ||
| + | style='mso-font-width:0%'><span style='mso-list:Ignore'>-<span | ||
| + | style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>FACs</span> results: New transformations | ||
| + | didn’t work. The control didn’t work.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 11: <o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Set up <span class=SpellE>FACs</span> on new transformation of: | ||
| + | <o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>- Silenced strain Adh-LexA-Sir2/Cyc-5’-GFP (Medium: SD-<span | ||
| + | class=SpellE>Leu</span> +0.5% <span class=SpellE>Glu</span>)<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span> <o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | MC-Esa1 <o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Cyc</span>- <span | ||
| + | class=SpellE>LexA</span> FL-Esa1<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal>- Silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP (Medium: <span | ||
| + | class=SpellE>SRaf</span> Comp +2% Gal)<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Cyc</span>- <span | ||
| + | class=SpellE>LexA</span> FL-Esa1<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>FACs</span> results: Too much background - | ||
| + | unclear results<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>AarI</span> digestion of VP64 - run gel - | ||
| + | couldn’t cut and gel purify VP64<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 12:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Digest Fig-<span class=SpellE>LexA</span> FL-<span | ||
| + | class=SpellE>mCherry</span> & Fig-<span class=SpellE>LexA</span> MC-<span | ||
| + | class=SpellE>mCherry</span> with <span class=SpellE>PspOMI</span> and <span | ||
| + | class=SpellE>SacI</span><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Ligation</span> into 2m plasmid:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Fig-<span class=SpellE>LexA</span> | ||
| + | MC-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal>Set up <span class=SpellE>FACs</span> on:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | MC-Esa1<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | FL-Esa1<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | DBD-Esa1<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span class=SpellE>Adh-LexA</span> | ||
| + | FL-<span class=SpellE>mCherry</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>FACs</span> results: control wasn’t | ||
| + | perfect - <span class=SpellE><span class=GramE>Adh-</span>LexA</span> MC-Esa1 | ||
| + | and <span class=SpellE>Adh-LexA</span> FL-<span class=SpellE>mCherry</span> | ||
| + | seems to be working.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 13:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Run Western Blot on prepared samples of <o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=GramE>for</span> detecting <span class=SpellE>LexA</span> | ||
| + | and <span class=SpellE>Hexokinase</span>. <o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Result - <u>partial success</u> - western blot worked - <span | ||
| + | class=SpellE>hexokinase</span> showed up, but the <span class=SpellE>LexA</span> | ||
| + | protein wasn’t detected.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 14 – 18:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Repeat western blot <span style='font-family:Wingdings; | ||
| + | mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; | ||
| + | mso-char-type:symbol;mso-symbol-font-family:Wingdings'><span style='mso-char-type: | ||
| + | symbol;mso-symbol-font-family:Wingdings'>à</span></span> gel failed to transfer | ||
| + | to the paper.<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Prepare new Quick <span class=SpellE>Lysates</span> for | ||
| + | Western Blot of:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR><span style='mso-fareast-language: | ||
| + | ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain </span>Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 19:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Testing the shuttle vectors from AO<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span style='mso-tab-count:1'> </span>Andrew has | ||
| + | done:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l0 level1 lfo2; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>1)<span | ||
| + | style='font:7.0pt "Times New Roman"'> </span></span><![endif]><span | ||
| + | dir=LTR>Cut AO shuttle vectors with <span class=SpellE>AarI</span>, <span | ||
| + | class=SpellE>XmaI</span> digested, CIP treated, gel purified.<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l0 level1 lfo2; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>2)<span | ||
| + | style='font:7.0pt "Times New Roman"'> </span></span><![endif]><span | ||
| + | dir=LTR>Cut AB and BD donors with <span class=SpellE>AarI</span>, gel purified.<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l0 level1 lfo2; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>3)<span | ||
| + | style='font:7.0pt "Times New Roman"'> </span></span><![endif]><span | ||
| + | dir=LTR><span class=SpellE>Ligations</span><o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Plate 1: AB shuttle + AB <span | ||
| + | class=SpellE>LacI</span> - 1107bp<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Plate 2: AB shuttle + <span | ||
| + | class=SpellE>LexA</span> (1-261) - 261bp (Probably FAILED)<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Plate 3: BD shuttle + RPD3cat - | ||
| + | 1000bp<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; | ||
| + | tab-stops:list .75in'><![if !supportLists]><span style='mso-font-width:0%'><span | ||
| + | style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> | ||
| + | </span></span></span><![endif]><span dir=LTR>Plate 4: BD shuttle + <span | ||
| + | class=SpellE>LexA</span> (1-261) - 261bp<o:p></o:p></span></p> | ||
| + | |||
| + | <p class=MsoNormal>I continued work:<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><span class=SpellE>Miniprep</span> Plates 1-4<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Digest vectors with <span class=SpellE>EcoRI</span> & <span | ||
| + | class=SpellE>SpeI</span> - Run gel - digestion failed<o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal>Repeat digestion with <span class=SpellE>EcoRI</span> & <span | ||
| + | class=SpellE>SpeI</span> - Run gel – <u>success<o:p></o:p></u></p> | ||
| + | |||
| + | <p class=MsoNormal><u><o:p><span style='text-decoration:none'> </span></o:p></u></p> | ||
| + | |||
| + | <p class=MsoNormal><b><u><span style='font-size:14.0pt;font-family:"Segoe Script"'>Week | ||
| + | 20:<o:p></o:p></span></u></b></p> | ||
| + | |||
| + | <p class=MsoNormal>Run the final gel that shows <span class=SpellE>AarI</span> | ||
| + | cut vectors and <span class=SpellE>EcoRI</span> & <span class=SpellE>SpeI</span> | ||
| + | Shuttle vectors - <u>success</u><o:p></o:p></p> | ||
| + | |||
| + | <p class=MsoNormal><o:p> </o:p></p> | ||
| + | |||
| + | </div> | ||
| + | |||
| + | </body> | ||
| + | |||
| + | </html> | ||
Latest revision as of 07:26, 22 October 2008
Leeza Sergeeva
Notebook 6/18/08 – 10/24/08
Milestones:
Objective:
Disrupt silencing to activate a gene.
Remodel chromatin so that heterochromatin can be uncoiled
and opened into euchromatin so they can be activated and freely available.
We’re turning on
the already silenced genes to produce the desired response (shift the activated
genes towards silencing) by opening heterochromatin and acetylating them to
become euchromatin and open.
Rationale - In
some cases, we will want differentiation to lead to gene expression (instead of
gene silencing).
Milestone: Create multiple fusions of DNA binding
domains and activations domains.
More specifically…
We are creating our anti-silencing circuits using various
DNA Binding Domains and activation domains. (As shown below)
|
|
DNA Binding Domains |
||
|
Activation domains | |
LexA PEG 202 (Full Length) NLS |
LexA 1-261 (DBD-short length)
NLS |
LexA 1-261 mCherry
NLS |
|
Sas2 |
+ |
+ |
+ |
|
Esa1 |
+ |
+ |
+ |
|
VP64 |
|
|
|
|
mCherry |
+ |
|
+ |
(+ - indicates what I’ve finally got)
The function of each is such:
1. We’re using various LexA domains to compete for the LexA binding
site. This won’t necessarily open heterochromatin, but it will prevent DNA from
becoming heterochromatin because it will block and HDAC from binding to the LexA binding site and therefore inhibit the DNA from
becoming silenced.
2. We’re using HATs such as Sas2
and Esa1 because they will cause an oozing model where we end up acetylating
the heterochromatin and “open” it into euchromatin.
3. We’re using VP64 - as a reader to attract HATs (writers) to they will acetylate
for us and open the heterochromatin for us indirectly.
4. We’re using mCherry just as a
reporter of expression level so we can run a FACs on
it.
Actual work done:
Week
1-2:
Sequencing VP64
PCR VP64
Week
3:
TOPO clone VP64
Digest PCR product of VP64 with EcoRI
- run gel - VP64 failed
Bacterial transformation of pFig1
Miniprep more of p315CYC
AarI digestion of p315CYC
Week
4:
Run gel to check if AarI digestion
of p315CYC worked (success)
TOPO clone mCherry
Miniprep mCherry
Sequence mCherry
Week
5:
EcoRI Diagnostic Digest of mCherry
AarI digestion of LexA DBD, LexA FL, LexA mCherry,p315ADH, p305GAL and
VP16
Run gel on AarI digestion
(success)
Gel purify LexA DBD, LexA FL, LexA mCherry,
p315ADH, p305GAL and VP16
Ligations - failed
Cyc-LexA DBD; Cyc-LexA FL; Cyc-mCherry; Cyc–VP16
Gal-LexA DBD; Gal-LexA FL; Gal-mCherry; Gal-VP16
AarI digestion of LexA DBD, VP16, LexA mCherry, Sas2, pGal, pAdh, pCyc.
Rung gel - digestion failed
Ligations - success
-
Gal-LexADBD-VP16
-
Gal-LexA
FL-VP16
-
Gal-LexA
mCherry-VP16
Bacterial Transformation of successful ligations
Week
6:
AarI digestion of VP16, Esa1, pGal, pCyc, Sas2, LexA mCherry.
Run Gel - VP16 (failed)
Colony PCR of ligations
-
Gal-LexADBD-VP16 Success
-
Gal-LexA
FL-VP16 Success
-
Gal-LexA
mCherry-VP16 Success
Yeast transformation of successful ligations
into silenced strains:
-
Adh-LexA-Sir2 / Cyc-5’-GFP
-
Cyc-LexA-Sir2 / Cyc-5’-GFP
Ligations - success
|
Gal1-LexAmCherry-Esa1 |
|
Gal1-LexAmCherry-Sas2 |
|
Gal1-LexA PEG 202 (FL)-Esa1 |
|
Gal1-LexA PEG 202 (FL)-Sas2 |
|
Gal1-LexA DBD-Esa1 |
|
Gal1-LexA DBD-Sas2 |
|
Adh1-LexAmCherry-Esa1 |
|
Adh1-LexAmCherry-Sas2 |
|
Adh1-LexA PEG 202 (FL)-Esa1 |
|
Adh1-LexA PEG 202 (FL)-Sas2 |
|
Adh1-LexA DBD-Esa1 |
|
Adh1-LexA DBD-Sas2 |
|
Cyc1-LexAmCherry-Esa1 |
|
Cyc1-LexAmCherry-Sas2 |
|
Cyc1-LexA PEG 202 (FL)-Esa1 |
|
Cyc1-LexA PEG 202 (FL)-Sas2 |
|
Cyc1-LexA DBD-Esa1 |
|
Cyc1-LexA DBD-Sas2 |
Week
7:
Cut p305Gal with XcmI
Bacterial Transformation of ligations
Colonies PCR- run gel - unclear
results
Ligations – success - Bacterial
Transformations - success
|
Fig1-LexAmCherry-Esa1 |
|
Fig1-LexAmCherry-Sas2 |
|
Fig1-LexA PEG 202 (FL)-Esa1 |
|
Fig1-LexA PEG 202 (FL)-Sas2 |
|
Fig1-LexA DBD-Esa1 |
|
Fig1-LexA DBD-Sas2 |
Miniprep colonies of Gal, Adh and Cyc
ligations
Diagnostic digest of Gal, Adh and Cyc ligations with PspOMI & SacI - success
Miniprep Fig ligations
Diagnostic digest of Fig ligations
with XhoI & NotI - success
Repeat of diagnostic digest of Gal, Adh and Cyc ligations with PspOMI & SacI - success
AarI digestions of LexA FL, ADH, VP16, mCherry
Run Gel - LexA
FL and ADH failed
Week
8:
Yeast transformation of Gal ligations
into silenced strains:
|
Gal1-LexA-Sir2 / Cyc-GFP |
|
Adh1-LexA-Sir2 / Cyc-5'opr-GFP |
|
Gal10-LexA-Sir2 / Cyc-GFP |
|
Cyc1-LexA-Sir2 / Cyc-5'opr-GFP |
Diagnostic digest of Gal Ligations
with XhoI & NotI - success
Restrick Gal yeasts on SRaf + 2% Gal and SD-Leu
Week
9:
Prepare Gal yeasts for FACs in SD
+ 2% Gal and SD-Leu medium.
Successful FACs
results that shows unsilencing of silenced
gene:
-
Gal-LexA
FL-EsaI on Gal10-LexA-Sir2 / Cyc-GFP
-
Gal-LexA
FL-Sas2 on Gal10-LexA-Sir2 / Cyc-GFP
-
Gal-LexA
MC-EsaI on Gal10-LexA-Sir2 / Cyc-GFP
-
Gal-LexA
FL-EsaI on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP
-
Gal-LexA
MC-EsaI on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP
Prepare successful FACs results
for repeat FACs experiment in –gal and +gal medium. (SRaf +2% gal and SDComp)
Successful FACs results
that show strong unsilencing:
-
Gal-LexA
MC-EsaI on Gal10-LexA-Sir2 / Cyc-GFP
-
Gal-LexA
FL-Sas2 on Gal10-LexA-Sir2 / Cyc-GFP
Set up FACs experiment unsilencing strain on silencing strain
Adh-LexA-Sir2/Cyc-5’-GFP:
|
Adh1-LexAmCherry-Esa1 |
|
Adh1-LexAmCherry-Sas2 |
|
Cyc1-LexA PEG 202
(FL)-Esa1 |
|
Cyc1-LexA PEG 202
(FL)-Sas2 |
|
Adh1-LexA
DBD-Esa1 |
|
Adh1-LexA
DBD-Sas2 |
|
Fig1-LexAmCherry-Esa1 |
|
Fig1-LexAmCherry-Sas2 |
|
Fig1-LexA PEG 202
(FL)-Esa1 |
|
Fig1-LexA PEG 202
(FL)-Sas2 |
|
Adh1-LexA PEG 202
(FL)-Esa1 |
|
Adh1-LexA PEG 202
(FL)-Sas2 |
FACs results:
The control strain (silenced strain wasn’t fully silenced)
Discovery - The low concentration of EsaI induces silencing, but the high concentration
unsilence the silenced gene.
Set up cultures for FACs :
-
Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
-
Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
-
Gal-LexA
PEG 202 (FL)-Esa1 on silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP
FACs results - success - unsilenced silenced genes
Week
10:
Transform Adh1-LexAmCherry-Esa1
(wrong plasmid) and
Cyc1-LexA PEG 202 (FL)-Esa1 again into yeast silenced strain
Adh-LexA-Sir2/Cyc-5’-GFP. (FAILURE!!! MISTAKE!!! TRANSFORMED WRONG PLASMID!!!)
Prepare Quick Lysates for Western
Blot of:
-
Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
-
Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
LigationsàBacterial
transformations - partial success
-
Adh-LexA
FL-mCherry
-
Adh-LexA
FL-VP64
-
Adh-LexA
MC-mCherry
-
Adh-LexA
DBD-VP64
-
Gal-LexA
FL-mCherry
-
Gal-LexA
FL-VP64
-
Gal-LexA
MC- mCherry
-
Gal-LexA
DBD-VP64
-
Fig-LexA
FL-mCherry
-
Fig-LexA
FL-VP64
-
Fig-LexA
MC- mCherry
-
Fig-LexA
DBD-VP64
Miniprep succeeded ligations
Cut Gal vectors with XcmI (for
yeast transformation)
Yeast Transformation of succeeded ligations
into new silenced strains (from Alex):
-
Adh-LexA-Sir2 / Cyc-5’-GFP
-
Cyc-LexA-Sir2 / Cyc-5’-GFP
Diagnostic Digest of succeeded ligations
with PspOMI & SacI - partial
success
Run gel - worked:
-
Adh-LexA
FL-mCherry
-
Gal-LexA
FL-mCherry
-
Gal-LexA
MC- mCherry
-
Fig-LexA
FL-mCherry
-
Fig-LexA
MC- mCherry
Set up FACs experiment on
-
Adh1-LexA MC-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
-
Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
FACs results: New transformations
didn’t work. The control didn’t work.
Week
11:
Set up FACs on new transformation of:
- Silenced strain Adh-LexA-Sir2/Cyc-5’-GFP (Medium: SD-Leu +0.5% Glu)
-
Adh-LexA
FL-mCherry
-
Adh-LexA
MC-Esa1
-
Cyc- LexA FL-Esa1
- Silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP (Medium: SRaf Comp +2% Gal)
-
Adh-LexA
FL-mCherry
-
Cyc- LexA FL-Esa1
FACs results: Too much background -
unclear results
AarI digestion of VP64 - run gel -
couldn’t cut and gel purify VP64
Week
12:
Digest Fig-LexA FL-mCherry & Fig-LexA MC-mCherry with PspOMI and SacI
Ligation into 2m plasmid:
-
Fig-LexA
FL-mCherry
-
Fig-LexA
MC-mCherry
Set up FACs on:
-
Adh-LexA
MC-Esa1
-
Adh-LexA
FL-Esa1
-
Adh-LexA
DBD-Esa1
-
Adh-LexA
FL-mCherry
FACs results: control wasn’t
perfect - Adh-LexA MC-Esa1
and Adh-LexA FL-mCherry
seems to be working.
Week
13:
Run Western Blot on prepared samples of
-
Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
-
Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
for detecting LexA
and Hexokinase.
Result - partial success - western blot worked - hexokinase showed up, but the LexA
protein wasn’t detected.
Week
14 – 18:
Repeat western blot à gel failed to transfer
to the paper.
Prepare new Quick Lysates for
Western Blot of:
-
Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
-
Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP
Week
19:
Testing the shuttle vectors from AO
Andrew has
done:
1) Cut AO shuttle vectors with AarI, XmaI digested, CIP treated, gel purified.
2) Cut AB and BD donors with AarI, gel purified.
3) Ligations
-
Plate 1: AB shuttle + AB LacI - 1107bp
-
Plate 2: AB shuttle + LexA (1-261) - 261bp (Probably FAILED)
-
Plate 3: BD shuttle + RPD3cat -
1000bp
-
Plate 4: BD shuttle + LexA (1-261) - 261bp
I continued work:
Miniprep Plates 1-4
Digest vectors with EcoRI & SpeI - Run gel - digestion failed
Repeat digestion with EcoRI & SpeI - Run gel – success
Week
20:
Run the final gel that shows AarI
cut vectors and EcoRI & SpeI
Shuttle vectors - success
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