BISC 219/F10: RNAi Lab 9 - Revision history

BISC 219/F10: RNAi Lab 9 - Revision history http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&action=history Revision history for this page on the wiki en MediaWiki 1.13.2 Sun, 26 May 2013 08:33:12 GMT Melissa Beers at 14:03, 18 May 2011 http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=509335&oldid=prev <p></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 14:03, 18 May 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 81:</td> <td colspan="2" class="diff-lineno">Line 81:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Assignment ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Assignment ==</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.</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><ins class="diffchange diffchange-inline"><div class=noprint></ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Links to Labs& Project Info==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Links to Labs& Project Info==</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Series1:<BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Series1:<BR></div></td></tr> <tr><td colspan="2" class="diff-lineno">Line 106:</td> <td colspan="2" class="diff-lineno">Line 106:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[BISC 219/F10: RNAi Lab 10 | Lab 10: Scoring your worms and RNA purification]]<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[BISC 219/F10: RNAi Lab 10 | Lab 10: Scoring your worms and RNA purification]]<br></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[BISC 219/F10: RNAi Lab 11 | Lab 11: RT PCR reactions]]<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[BISC 219/F10: RNAi Lab 11 | Lab 11: RT PCR reactions]]<br><br></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></div></ins></div></td></tr>  </table> Wed, 18 May 2011 14:03:49 GMT Melissa Beers http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9 Melissa Beers at 19:44, 9 December 2010 http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=478728&oldid=prev <p></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 19:44, 9 December 2010</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 24:</td> <td colspan="2" class="diff-lineno">Line 24:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center>[[Image:L4440.tif]]</center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center>[[Image:L4440.tif]]</center></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>More specifically: The bacteria cells of strain HTll5(<del class="diffchange diffchange-inline">DE</del>) contain the T7 RNA polymerase gene (contained within a stable insertion of a modified lambda prophage λ DE3) under the control of lac operon regulatory elements.  This allows expression of T7 polymerase to be controlled by isopropyl-β-D-thiogalactopyranoside (IPTG), a lactose analogue that induces expression of genes under the control of the lac operon o gene.  When IPTG is added, the cells will begin to synthesize lots of T7 RNA polymerase.  This T7 RNA polymerase can then bind to the T7 promoter sites on the plasmid and begin to synthesize RNA from both T7 RNA polymerase sites.  Because the two single strands of RNA are complementary to each other they will form double stranded RNA within the bacterial cell.  The IPTG induction allows us to "turn on" and express the plasmid gene of interest only when we want to and it allow us to make much higher levels of RNA for RNA interference than would be made without this induction.  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>More specifically: The bacteria cells of strain HTll5(<ins class="diffchange diffchange-inline">DE3</ins>) contain the T7 RNA polymerase gene (contained within a stable insertion of a modified lambda prophage λ DE3) under the control of lac operon regulatory elements.  This allows expression of T7 polymerase to be controlled by isopropyl-β-D-thiogalactopyranoside (IPTG), a lactose analogue that induces expression of genes under the control of the lac operon o gene.  When IPTG is added, the cells will begin to synthesize lots of T7 RNA polymerase.  This T7 RNA polymerase can then bind to the T7 promoter sites on the plasmid and begin to synthesize RNA from both T7 RNA polymerase sites.  Because the two single strands of RNA are complementary to each other they will form double stranded RNA within the bacterial cell.  The IPTG induction allows us to "turn on" and express the plasmid gene of interest only when we want to and it allow us to make much higher levels of RNA for RNA interference than would be made without this induction.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Another useful thing about E coli strain HT 115(<del class="diffchange diffchange-inline">DE</del>)is that this particular strain is '''deficient''' for the RNAaseIII enzyme that degrades double stranded RNA (dsRNA) in the bacterial cell. This allows for the accumulation of dsRNA in the cell and, thus, our ability to induce and RNAi effect!  This ''E. coli'' strain carries a tetracyclin resistance gene so these cells can be selected on media containing tetracyclin, while the plasmid contains an ampicillin resistance gene that allows only transfomed cells to grow on media containing ampicillin.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Another useful thing about E coli strain HT 115(<ins class="diffchange diffchange-inline">DE3</ins>)is that this particular strain is '''deficient''' for the RNAaseIII enzyme that degrades double stranded RNA (dsRNA) in the bacterial cell. This allows for the accumulation of dsRNA in the cell and, thus, our ability to induce and RNAi effect!  This ''E. coli'' strain carries a tetracyclin resistance gene so these cells can be selected on media containing tetracyclin, while the plasmid contains an ampicillin resistance gene that allows only transfomed cells to grow on media containing ampicillin.<br></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our goal is to upregulate production of dsRNA of our worm gene of interest in pL4440 vector plasmids containing that gene in HT115(<del class="diffchange diffchange-inline">DE</del>) bacteria. <br><bR></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our goal is to upregulate production of dsRNA of our worm gene of interest in pL4440 vector plasmids containing that gene in HT115(<ins class="diffchange diffchange-inline">DE3</ins>) bacteria. <br><bR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''To induce your cultures:''' <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''To induce your cultures:''' <br></div></td></tr>  </table> Thu, 09 Dec 2010 19:44:18 GMT Melissa Beers http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9 Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */ http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=475791&oldid=prev <p><span class="autocomment">Outline of Experimental Design for REVERSE Genetics Project</span></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 16:29, 29 November 2010</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 76:</td> <td colspan="2" class="diff-lineno">Line 76:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Isolate RNA from RNAi worms and control worms of same strains.<BR><BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Isolate RNA from RNAi worms and control worms of same strains.<BR><BR></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest <del class="diffchange diffchange-inline">and check its size</del>, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare size of amplified DNA to known size of coding regions gene of interest.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare size of amplified DNA to known size of coding regions gene of interest.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>  </table> Mon, 29 Nov 2010 16:29:34 GMT Tucker Crum http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9 Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */ http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=474304&oldid=prev <p><span class="autocomment">Outline of Experimental Design for REVERSE Genetics Project</span></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 21:54, 23 November 2010</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 77:</td> <td colspan="2" class="diff-lineno">Line 77:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Isolate RNA from RNAi worms and control worms of same strains.<BR><BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Isolate RNA from RNAi worms and control worms of same strains.<BR><BR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest and check its size, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest and check its size, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare size of amplified <del class="diffchange diffchange-inline">fragment </del>to known size of <del class="diffchange diffchange-inline"> </del>coding regions <del class="diffchange diffchange-inline">of </del>gene of interest.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare size of amplified <ins class="diffchange diffchange-inline">DNA </ins>to known size of coding regions gene of interest.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Assignment ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Assignment ==</div></td></tr>  </table> Tue, 23 Nov 2010 21:54:45 GMT Tucker Crum http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9 Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */ http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=474303&oldid=prev <p><span class="autocomment">Outline of Experimental Design for REVERSE Genetics Project</span></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 21:53, 23 November 2010</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 76:</td> <td colspan="2" class="diff-lineno">Line 76:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Isolate RNA from RNAi worms and control worms of same strains.<BR><BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Isolate RNA from RNAi worms and control worms of same strains.<BR><BR></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest and check its size, using worm RNA and then cDNA as template<del class="diffchange diffchange-inline">, </del>isolated from treated RNAi worms and untreated worms of the same species.<BR><BR></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest and check its size, using worm RNA and then cDNA as template<ins class="diffchange diffchange-inline">. The RNA is </ins>isolated from treated RNAi worms and untreated worms of the same species.<BR><BR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare size of amplified fragment to known size of  coding regions of gene of interest.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare size of amplified fragment to known size of  coding regions of gene of interest.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>  </table> Tue, 23 Nov 2010 21:53:30 GMT Tucker Crum http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9 Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */ http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=474297&oldid=prev <p><span class="autocomment">Outline of Experimental Design for REVERSE Genetics Project</span></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 21:02, 23 November 2010</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 68:</td> <td colspan="2" class="diff-lineno">Line 68:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Culture the selected colony from colony pcr to create a lot of copies of these bacteria</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Culture the selected colony from colony pcr to create a lot of copies of these bacteria</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Isolate the cloned plasmid DNA from that cultured colony by miniprep;<BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Isolate the cloned plasmid DNA from that cultured colony by miniprep;<BR></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Retransform isolated plasmids (with gene interest) into <del class="diffchange diffchange-inline">ITPG inducable </del>HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;<BR></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;<BR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Select for transformants on media with ampicillin  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Select for transformants on media with ampicillin  </div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Choose an isolated colony to culture and make lots of bacteria;  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Choose an isolated colony to culture and make lots of <ins class="diffchange diffchange-inline">feeder strain </ins>bacteria; <ins class="diffchange diffchange-inline"><br></ins></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Induce expression of ''C. elegans'' gene dsRNA from pL4440 vector by IPTG induction <del class="diffchange diffchange-inline">of log phase culture of HT115(DE) ''E</del>. <del class="diffchange diffchange-inline">coli''</del><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Induce expression of ''C. elegans'' gene dsRNA from <ins class="diffchange diffchange-inline">the </ins>pL4440 vector <ins class="diffchange diffchange-inline">in the bacteria </ins>by IPTG induction. <br></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Seed NM lite worm growth media plates with <del class="diffchange diffchange-inline">induced </del>feeder strain <del class="diffchange diffchange-inline">bacteria</del><BR>  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Seed NM lite worm growth media plates with feeder strain <ins class="diffchange diffchange-inline">produced as described </ins><BR>  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that <del class="diffchange diffchange-inline">we </del>NOT fed feeder strain bacteria.<BR><BR></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that <ins class="diffchange diffchange-inline">were </ins>NOT fed feeder strain bacteria.<BR><BR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Isolate RNA from RNAi worms and control worms of same strains.<BR><BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Isolate RNA from RNAi worms and control worms of same strains.<BR><BR></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Perform RT-PCR (Reverse Transcriptase) <del class="diffchange diffchange-inline">using the mRNA of </del>the gene of interest as template, isolated from <del class="diffchange diffchange-inline">the </del>RNAi worms.<BR><BR></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Perform RT-PCR (Reverse Transcriptase)<ins class="diffchange diffchange-inline">to amplify </ins>the <ins class="diffchange diffchange-inline">''C. elegans'' </ins>gene of interest <ins class="diffchange diffchange-inline">and check its size, using worm RNA and then cDNA </ins>as template, isolated from <ins class="diffchange diffchange-inline">treated </ins>RNAi worms <ins class="diffchange diffchange-inline">and untreated worms of the same species</ins>.<BR><BR></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Visualize <del class="diffchange diffchange-inline">cDNA </del>in the pcr product by agarose gel electrophoresis and compare size of amplified fragment to known size of  coding regions of gene of interest.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Visualize <ins class="diffchange diffchange-inline">the worm gene of interst </ins>in the pcr product by agarose gel electrophoresis and compare size of amplified fragment to known size of  coding regions of gene of interest.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Assignment ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Assignment ==</div></td></tr>  </table> Tue, 23 Nov 2010 21:02:15 GMT Tucker Crum http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9 Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */ http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=471787&oldid=prev <p><span class="autocomment">Outline of Experimental Design for REVERSE Genetics Project</span></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 17:02, 12 November 2010</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 63:</td> <td colspan="2" class="diff-lineno">Line 63:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Clean up DNA (remove enzymes); <BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Clean up DNA (remove enzymes); <BR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Cloning: ligate gene into vector plasmid with amp resistance gene ;<BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Cloning: ligate gene into vector plasmid with amp resistance gene ;<BR></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Transform competent bacterial cells <del class="diffchange diffchange-inline">of a strain genetically modified to be tetracycline resistant</del>;  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Transform competent bacterial cells;  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Select for transformants on media with ampicillin;<BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Select for transformants on media with ampicillin;<BR></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst</div></td></tr>  </table> Fri, 12 Nov 2010 17:02:21 GMT Tucker Crum http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9 Tucker Crum: /* Lab 9: Series 3 Reverse Genetics-Induction of HT115(DE3) feeding strains for RNAi */ http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=471770&oldid=prev <p><span class="autocomment">Lab 9: Series 3 Reverse Genetics-Induction of HT115(DE3) feeding strains for RNAi</span></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 14:52, 12 November 2010</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 52:</td> <td colspan="2" class="diff-lineno">Line 52:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#On 1 of the control plates add 2 L4 wild type (N2) hermaphrodites</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#On 1 of the control plates add 2 L4 wild type (N2) hermaphrodites</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#On 1 of the control plates add 2 L4 ''rrf-3'' hermaphrodites</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#On 1 of the control plates add 2 L4 ''rrf-3'' hermaphrodites</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Wrap <del class="diffchange diffchange-inline">all of your </del>plates <del class="diffchange diffchange-inline">in an elastic </del>and <del class="diffchange diffchange-inline">stick </del>in your lab day box in the worm incubator set at 23°C<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Wrap <ins class="diffchange diffchange-inline">the 3 </ins>plates <ins class="diffchange diffchange-inline">of each strain (N2 or ''rrf-3'') with elastics </ins>and <ins class="diffchange diffchange-inline">incubate the N2 worms </ins>in your lab day box in the worm incubator set at 23°C<ins class="diffchange diffchange-inline">. Incubate the 3 plates of ''rrf-3'' strain worms at room temperature in your lab day project box in the stock area. '''DO NOT incubate the rrf-3 worms at 23C!!!!'''</ins><br></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>You will score your phenotypes in the next lab.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>You will score your phenotypes in the next lab.</div></td></tr>  </table> Fri, 12 Nov 2010 14:52:58 GMT Tucker Crum http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9 Tucker Crum: /* Lab 9: Series 3 Reverse Genetics-Induction of HT115(DE3) feeding strains for RNAi */ http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=470422&oldid=prev <p><span class="autocomment">Lab 9: Series 3 Reverse Genetics-Induction of HT115(DE3) feeding strains for RNAi</span></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 14:59, 4 November 2010</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 32:</td> <td colspan="2" class="diff-lineno">Line 32:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''To induce your cultures:''' <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''To induce your cultures:''' <br></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Add 5 μL of 0.5 M IPTG to your culture.  What is the effective concentration of IPTG in your culture?</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Add 5 μL of 0.5 M IPTG to your culture.  What is the effective concentration of IPTG in your culture?</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Put your culture back in the 37°C incubator in the spinning wheel for approximately 3 hours.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Put your culture back in the 37°C incubator in the spinning wheel for approximately 3<ins class="diffchange diffchange-inline">-4 </ins>hours.</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#After the lab introduction, we will head <del class="diffchange diffchange-inline">up </del>to <del class="diffchange diffchange-inline">the penthouse </del>to discuss the papers you were assigned.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#After the lab introduction, we will head to <ins class="diffchange diffchange-inline">a more comfortable room </ins>to discuss the papers you were assigned.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''To do after induction is complete:'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''To do after induction is complete:'''</div></td></tr>  </table> Thu, 04 Nov 2010 14:59:49 GMT Tucker Crum http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9 Tucker Crum: /* Lab 9: Series 3 Reverse Genetics-Induction of HT115(DE3) feeding strains for RNAi */ http://www.openwetware.org/index.php?title=BISC_219/F10:_RNAi_Lab_9&diff=470420&oldid=prev <p><span class="autocomment">Lab 9: Series 3 Reverse Genetics-Induction of HT115(DE3) feeding strains for RNAi</span></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 14:58, 4 November 2010</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 17:</td> <td colspan="2" class="diff-lineno">Line 17:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Your instructor or the lab staff will come in early in the morning and '''sub-culture''' your bacterial overnight.  The cells will be in stationary phase in the morning and sucessful induction requires log phase growth.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Your instructor or the lab staff will come in early in the morning and '''sub-culture''' your bacterial overnight.  The cells will be in stationary phase in the morning and sucessful induction requires log phase growth.<br></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To create the subculture of bacteria your cultures will be diluted 1:<del class="diffchange diffchange-inline">10 </del>(<del class="diffchange diffchange-inline">500 μL </del>of culture into 4<del class="diffchange diffchange-inline">.5 </del>ml of LB + amp).  These cultures will be allowed to grow until lab time - approximately 3-4 hours.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To create the subculture of bacteria your cultures will be diluted 1:<ins class="diffchange diffchange-inline">5 </ins>(<ins class="diffchange diffchange-inline">1 mL </ins>of culture into 4 ml of LB + amp).  These cultures will be allowed to grow until lab time - approximately 3-4 hours.<br></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>When you come in to lab you will induce your cultures to make lots of dsRNA by adding IPTG to the culture and letting it continue to incubate for a few hours so the cell is full of dsRNA.  The IPTG will compete with the repressors on the lac o promoter and remove them and allow the gene for T7 RNA polymerase to be transcribed and then translated into the RNA polymerase protein.  The T7 RNA polymerase then binds to the T7 promoters on the pL4440 plasmid and transcribes our ''C. elegans'' DNA into RNA!  <BR><BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>When you come in to lab you will induce your cultures to make lots of dsRNA by adding IPTG to the culture and letting it continue to incubate for a few hours so the cell is full of dsRNA.  The IPTG will compete with the repressors on the lac o promoter and remove them and allow the gene for T7 RNA polymerase to be transcribed and then translated into the RNA polymerase protein.  The T7 RNA polymerase then binds to the T7 promoters on the pL4440 plasmid and transcribes our ''C. elegans'' DNA into RNA!  <BR><BR></div></td></tr>  </table> Thu, 04 Nov 2010 14:58:33 GMT Tucker Crum http://www.openwetware.org/wiki/Talk:BISC_219/F10:_RNAi_Lab_9