2020(S11) Lecture:week 2 - Revision history

http://www.openwetware.org/index.php?title=2020(S11)_Lecture:week_2&feed=atom&action=history 2020(S11) Lecture:week 2 - Revision history 2013-05-25T10:58:39Z Revision history for this page on the wiki MediaWiki 1.13.2 http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=492665&oldid=prev Nkuldell: /* The Polymerase Chain Reaction */ 2011-02-10T15:18:56Z

<p><span class="autocomment">The Polymerase Chain Reaction</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 15:18, 10 February 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 119:</td> <td colspan="2" class="diff-lineno">Line 119:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Here's the $64,000 question: are the restriction sites you chose for the pPRL or pGRN genes on that plasmid. If so, then YAY! that was easy! But if not, what do you do? Maybe choose a new plasmid and hope for a better outcome? Or maybe we can figure out a way to put the needed restriction sites onto the pPRL or pGRN genes. Look next to PCR</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Here's the $64,000 question: are the restriction sites you chose for the pPRL or pGRN genes on that plasmid. If so, then YAY! that was easy! But if not, what do you do? Maybe choose a new plasmid and hope for a better outcome? Or maybe we can figure out a way to put the needed restriction sites onto the pPRL or pGRN genes. Look next to PCR</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===The Polymerase Chain Reaction===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===The Polymerase Chain Reaction===</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">#</del>After a quick review for how this technique works and how it can be used to introduce special restriction sites, you'll have a chance to try it yourself.  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>After a quick review for how this technique works and how it can be used to introduce special restriction sites, you'll have a chance to try it yourself.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>====Option 1====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>====Option 1====</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Use a toothpick to mix one colony of either pPRL or pGRN from your transformation plate into 100 ul of water.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Use a toothpick to mix one colony of either pPRL or pGRN from your transformation plate into 100 ul of water.  </div></td></tr>  </table>

Nkuldell http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=492664&oldid=prev Nkuldell at 15:18, 10 February 2011 2011-02-10T15:18:30Z

<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 15:18, 10 February 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 115:</td> <td colspan="2" class="diff-lineno">Line 115:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># WOW! That's a lot of Gs, As, Ts, and Cs...definitely don't want to be looking for restriction sites the way we did for the paper versions of the gene. Luckily you can copy and paste the sequence into a search tool provided by NEB called [http://tools.neb.com/NEBcutter2/ NEB cutter.]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># WOW! That's a lot of Gs, As, Ts, and Cs...definitely don't want to be looking for restriction sites the way we did for the paper versions of the gene. Luckily you can copy and paste the sequence into a search tool provided by NEB called [http://tools.neb.com/NEBcutter2/ NEB cutter.]</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Having done that for your pPRL or pGRN sequence, can you find an enzyme that flanks the genes?</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Having done that for your pPRL or pGRN sequence, can you find an enzyme that flanks the genes?</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># OK...now you'll need someplace to put the gene. Go back to the <del class="diffchange diffchange-inline">[</del>[http://tools.neb.com/NEBcutter2/ NEB cutter] tool, clear any sequences that might be in the search box and then use the drop-down menu from the right tab to find a plasmid backbone. In "real life" you'd have some reason to choose one kind of destination plasmid over another, but for today you can randomly choose one to clone your purple expression cassette into.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># OK...now you'll need someplace to put the gene. Go back to the [http://tools.neb.com/NEBcutter2/ NEB cutter] tool, clear any sequences that might be in the search box and then use the drop-down menu from the right tab to find a plasmid backbone. In "real life" you'd have some reason to choose one kind of destination plasmid over another, but for today you can randomly choose one to clone your purple expression cassette into<ins class="diffchange diffchange-inline">.</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Once you press "submit" a drawing of a circular plasmid will show up on your screen and all the single cutter restriction sites will be labeled.</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Here's the $64,000 question: are the restriction sites you chose for the pPRL or pGRN genes on that plasmid. If so, then YAY! that was easy! But if not, what do you do? Maybe choose a new plasmid and hope for a better outcome? Or maybe we can figure out a way to put the needed restriction sites onto the pPRL or pGRN genes. Look next to PCR</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===The Polymerase Chain Reaction===</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#After a quick review for how this technique works and how it can be used to introduce special restriction sites, you'll have a chance to try it yourself. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">====Option 1====</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#Use a toothpick to mix one colony of either pPRL or pGRN from your transformation plate into 100 ul of water. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#Move 10 ul of that mixture to a tube that has 90 ul of PCR mix in it. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">====Option 2====</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#Add 10 ul of dilute DNA to a tube with 90 ul of PCR mix in it. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">====Next steps====</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">The reactions will be placed in a thermal cycler to heat and cool and hopefully make more of the DNA you need with the needed restriction sites on the ends. We'll come back to look at this DNA in a few weeks</ins>.</div></td></tr>  </table>

Nkuldell http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=492660&oldid=prev Nkuldell at 15:11, 10 February 2011 2011-02-10T15:11:00Z

<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 15:11, 10 February 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 115:</td> <td colspan="2" class="diff-lineno">Line 115:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># WOW! That's a lot of Gs, As, Ts, and Cs...definitely don't want to be looking for restriction sites the way we did for the paper versions of the gene. Luckily you can copy and paste the sequence into a search tool provided by NEB called [http://tools.neb.com/NEBcutter2/ NEB cutter.]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># WOW! That's a lot of Gs, As, Ts, and Cs...definitely don't want to be looking for restriction sites the way we did for the paper versions of the gene. Luckily you can copy and paste the sequence into a search tool provided by NEB called [http://tools.neb.com/NEBcutter2/ NEB cutter.]</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Having done that for your pPRL or pGRN sequence, can you find an enzyme that flanks the genes?</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Having done that for your pPRL or pGRN sequence, can you find an enzyme that flanks the genes?</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;"># OK...now you'll need someplace to put the gene. Go back to the [[http://tools.neb.com/NEBcutter2/ NEB cutter] tool, clear any sequences that might be in the search box and then use the drop-down menu from the right tab to find a plasmid backbone. In "real life" you'd have some reason to choose one kind of destination plasmid over another, but for today you can randomly choose one to clone your purple expression cassette into.</ins></div></td></tr>  </table>

Nkuldell http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=492649&oldid=prev Nkuldell at 11:22, 10 February 2011 2011-02-10T11:22:06Z

<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 11:22, 10 February 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 105:</td> <td colspan="2" class="diff-lineno">Line 105:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note 1:  the yellow circle must include the small gray box since that symbolizes elements needed for the plasmid to survive, e.g. the replication origin and the antibiotic marker.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note 1:  the yellow circle must include the small gray box since that symbolizes elements needed for the plasmid to survive, e.g. the replication origin and the antibiotic marker.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note 2: the blue sequence must contain all of the gene you are trying to clone. If you choose an enzyme that cuts inside of the gene then you will be losing information that is needed for its expression. Choose a different pair of enzymes if you can.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note 2: the blue sequence must contain all of the gene you are trying to clone. If you choose an enzyme that cuts inside of the gene then you will be losing information that is needed for its expression. Choose a different pair of enzymes if you can.  </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;">#*Note 3: were you able to "orient" your blue gene in the yellow plasmid or could it be cloned in either orientation?</ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Viola! You've cloned your gene...as least on paper.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Viola! You've cloned your gene...as least on paper.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===The main event: cloning pPRL or pGRN===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===The main event: cloning pPRL or pGRN===</div></td></tr> <tr><td colspan="2" class="diff-lineno">Line 112:</td> <td colspan="2" class="diff-lineno">Line 113:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Next, we'll find out what restriction sites are present at the ends of the pPRL and the pGRN gene clusters. To figure this out, let's use the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts.] In the homepage search bar type in the part number for either pPRL = BBa_K274002, or pGRN = BBa_K274004.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Next, we'll find out what restriction sites are present at the ends of the pPRL and the pGRN gene clusters. To figure this out, let's use the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts.] In the homepage search bar type in the part number for either pPRL = BBa_K274002, or pGRN = BBa_K274004.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Once you have been taken to the part page for those DNA functions, scroll down to the "sequence and features" diagram (shown above) and click the link on the right that says "Get selected sequence."</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Once you have been taken to the part page for those DNA functions, scroll down to the "sequence and features" diagram (shown above) and click the link on the right that says "Get selected sequence."</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">WOW! That's a lot of Gs, As, Ts, and Cs...definitely don't want to be looking for restriction sites the way we did for the paper versions of the gene. Luckily you can copy and paste the sequence into a search tool provided by NEB called [http://tools.neb.com/NEBcutter2/ NEB cutter.]</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Having done that for your pPRL or pGRN sequence, can you find an enzyme that flanks the genes?</ins></div></td></tr>  </table>

Nkuldell http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=492648&oldid=prev Nkuldell at 11:18, 10 February 2011 2011-02-10T11:18:36Z

<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 11:18, 10 February 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 108:</td> <td colspan="2" class="diff-lineno">Line 108:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===The main event: cloning pPRL or pGRN===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===The main event: cloning pPRL or pGRN===</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Let's now turn to a piece of DNA that you've been working with already, namely the genes that are cloned on the [http://partsregistry.org/Part:BBa_K274002 pPRL] and the [http://partsregistry.org/Part:BBa_K274004 pGRN] plasmids. To re-clone these, we'll need more than paper, scissors and tape. We'll need to know some real enzymes and we'll need some actual DNA. Enzymes can be found most easily at the website of a local company, [http://www.neb.com/nebecomm/default.asp New England Biolabs, NEB.]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Let's now turn to a piece of DNA that you've been working with already, namely the genes that are cloned on the [http://partsregistry.org/Part:BBa_K274002 pPRL] and the [http://partsregistry.org/Part:BBa_K274004 pGRN] plasmids. To re-clone these, we'll need more than paper, scissors and tape. We'll need to know some real enzymes and we'll need some actual DNA. Enzymes can be found most easily at the website of a local company, [http://www.neb.com/nebecomm/default.asp New England Biolabs, NEB.]</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;">[[Image:GetSequence 274002.png|thumb|right|500px| retrieving sequence info]] </ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Start by going to the [http://www.neb.com/nebecomm/default.asp NEB homepage] and typing in the DNA sequence for Enzyme 3, namely GGATCC. What is the identity of this enzyme? Repeat this for paper enzyme 6.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Start by going to the [http://www.neb.com/nebecomm/default.asp NEB homepage] and typing in the DNA sequence for Enzyme 3, namely GGATCC. What is the identity of this enzyme? Repeat this for paper enzyme 6.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Next, we'll find out what restriction sites are present at the ends of the pPRL and the pGRN gene clusters. To figure this out, let's use the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts.] In the homepage search bar type in the part number for either pPRL = BBa_K274002, or pGRN = BBa_K274004.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Next, we'll find out what restriction sites are present at the ends of the pPRL and the pGRN gene clusters. To figure this out, let's use the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts.] In the homepage search bar type in the part number for either pPRL = BBa_K274002, or pGRN = BBa_K274004.  </div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Once you have been taken to the part page for those DNA functions,</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Once you have been taken to the part page for those DNA functions, <ins class="diffchange diffchange-inline">scroll down to the "sequence and features" diagram (shown above) and click the link on the right that says "Get selected sequence."</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#</ins></div></td></tr>  </table>

Nkuldell http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=492646&oldid=prev Nkuldell at 11:14, 10 February 2011 2011-02-10T11:14:03Z

<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 11:14, 10 February 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 99:</td> <td colspan="2" class="diff-lineno">Line 99:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This exercise is adapted from [http://www.accessexcellence.org/RC/AB/WYW/wkbooks/SFTS/activity6.php access excellence.] You will need a yellow, blue and white page ahowing DNA sequences and enzyme recognition sites.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This exercise is adapted from [http://www.accessexcellence.org/RC/AB/WYW/wkbooks/SFTS/activity6.php access excellence.] You will need a yellow, blue and white page ahowing DNA sequences and enzyme recognition sites.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Begin by cutting the [[Media:Cloning gene blue.gif| blue page]] into strips and taping the strips together according to the numbers at the bottom of each strip. In the end you will have a line (not a circle) of paper, with a gray shaded section within it. This gray section is the gene you'd like to clone from some genomic DNA.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Begin by cutting the [[Media:Cloning gene blue.gif| blue page]] into strips and taping the strips together according to the numbers at the bottom of each strip. In the end you will have a line (not a circle) of paper, with a gray shaded section within it. This gray section is the gene you'd like to clone from some genomic DNA.</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Next scan this blue strip of paper for the [[<del class="diffchange diffchange-inline">Image</del>:Cloning enzymes.gif| enzyme restriction sites.]] In the interest of time today, please only identify on the blue strip of sequence where you can find the DNA recognition sequence for '''Enzyme 3, Enzyme 6 and Enzyme 9.''' When you find them, you can mark on the paper where the two strands of DNA would be divided by the enzyme, and also mark the enzyme number.  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Next scan this blue strip of paper for the [[<ins class="diffchange diffchange-inline">Media</ins>:Cloning enzymes.gif| enzyme restriction sites.]] In the interest of time today, please only identify on the blue strip of sequence where you can find the DNA recognition sequence for '''Enzyme 3, Enzyme 6 and Enzyme 9.''' When you find them, you can mark on the paper where the two strands of DNA would be divided by the enzyme, and also mark the enzyme number.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Next cut the [[Media:Cloning bkb yellow.gif| yellow page]] into the indicated strips, tape the strips together in any order, and finally tape the ends together to make a loop. This is the plasmid vector where you will "clone" your gene from the blue paper.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Next cut the [[Media:Cloning bkb yellow.gif| yellow page]] into the indicated strips, tape the strips together in any order, and finally tape the ends together to make a loop. This is the plasmid vector where you will "clone" your gene from the blue paper.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># On the yellow circle of sequence, find the recognition sites for Enzymes 3, 6 and 9.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># On the yellow circle of sequence, find the recognition sites for Enzymes 3, 6 and 9.  </div></td></tr> <tr><td colspan="2" class="diff-lineno">Line 105:</td> <td colspan="2" class="diff-lineno">Line 105:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note 1:  the yellow circle must include the small gray box since that symbolizes elements needed for the plasmid to survive, e.g. the replication origin and the antibiotic marker.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note 1:  the yellow circle must include the small gray box since that symbolizes elements needed for the plasmid to survive, e.g. the replication origin and the antibiotic marker.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note 2: the blue sequence must contain all of the gene you are trying to clone. If you choose an enzyme that cuts inside of the gene then you will be losing information that is needed for its expression. Choose a different pair of enzymes if you can.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note 2: the blue sequence must contain all of the gene you are trying to clone. If you choose an enzyme that cuts inside of the gene then you will be losing information that is needed for its expression. Choose a different pair of enzymes if you can.  </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;"># Viola! You've cloned your gene...as least on paper.</ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===The main event: cloning pPRL or pGRN===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===The main event: cloning pPRL or pGRN===</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;">Let's now turn to a piece of DNA that you've been working with already, namely the genes that are cloned on the [http://partsregistry.org/Part:BBa_K274002 pPRL] and the [http://partsregistry.org/Part:BBa_K274004 pGRN] plasmids. To re-clone these, we'll need more than paper, scissors and tape. We'll need to know some real enzymes and we'll need some actual DNA. Enzymes can be found most easily at the website of a local company, [http://www.neb.com/nebecomm/default.asp New England Biolabs, NEB.]</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"># Start by going to the [http://www.neb.com/nebecomm/default.asp NEB homepage] and typing in the DNA sequence for Enzyme 3, namely GGATCC. What is the identity of this enzyme? Repeat this for paper enzyme 6. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"># Next, we'll find out what restriction sites are present at the ends of the pPRL and the pGRN gene clusters. To figure this out, let's use the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts.] In the homepage search bar type in the part number for either pPRL = BBa_K274002, or pGRN = BBa_K274004. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Once you have been taken to the part page for those DNA functions,</ins></div></td></tr>  </table>

Nkuldell http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=492642&oldid=prev Nkuldell at 11:01, 10 February 2011 2011-02-10T11:01:07Z

<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 11:01, 10 February 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 98:</td> <td colspan="2" class="diff-lineno">Line 98:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Warm up: Restriction enzymes to clone a paper gene===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Warm up: Restriction enzymes to clone a paper gene===</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This exercise is adapted from [http://www.accessexcellence.org/RC/AB/WYW/wkbooks/SFTS/activity6.php access excellence.] You will need a yellow, blue and white page ahowing DNA sequences and enzyme recognition sites.  </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This exercise is adapted from [http://www.accessexcellence.org/RC/AB/WYW/wkbooks/SFTS/activity6.php access excellence.] You will need a yellow, blue and white page ahowing DNA sequences and enzyme recognition sites.  </div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Begin by cutting the blue page into strips and taping the strips together according to the numbers at the bottom of each strip. In the end you will have a line (not a circle) of paper, with a <del class="diffchange diffchange-inline">grey </del>shaded section within it. This <del class="diffchange diffchange-inline">grey </del>section is the gene you'd like to clone <del class="diffchange diffchange-inline">but on the </del>genomic DNA.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Begin by cutting the <ins class="diffchange diffchange-inline">[[Media:Cloning gene blue.gif| </ins>blue page<ins class="diffchange diffchange-inline">]] </ins>into strips and taping the strips together according to the numbers at the bottom of each strip. In the end you will have a line (not a circle) of paper, with a <ins class="diffchange diffchange-inline">gray </ins>shaded section within it. This <ins class="diffchange diffchange-inline">gray </ins>section is the gene you'd like to clone <ins class="diffchange diffchange-inline">from some </ins>genomic DNA.</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Next scan this blue strip of paper for the enzyme restriction sites. <del class="diffchange diffchange-inline">You are welcome to look for all the enzymes, but in </del>the interest of time today, please only identify on the <del class="diffchange diffchange-inline">paper </del>where you can find the DNA recognition sequence for '''Enzyme 3, Enzyme 6 and Enzyme 9.''' When you find them, you can mark on the paper</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Next scan this blue strip of paper for the <ins class="diffchange diffchange-inline">[[Image:Cloning enzymes.gif| </ins>enzyme restriction sites.<ins class="diffchange diffchange-inline">]] In </ins>the interest of time today, please only identify on the <ins class="diffchange diffchange-inline">blue strip of sequence </ins>where you can find the DNA recognition sequence for '''Enzyme 3, Enzyme 6 and Enzyme 9.''' When you find them, you can mark on the paper <ins class="diffchange diffchange-inline">where the two strands of DNA would be divided by the enzyme, and also mark the enzyme number. </ins></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">Next cut the [[Media:Cloning bkb yellow.gif| yellow page]] into the indicated strips, tape the strips together in any order, and finally tape the ends together to make a loop. This is the plasmid vector where you will "clone" your gene from the blue paper.</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># On the yellow circle of sequence, find the recognition sites for Enzymes 3, 6 and 9. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Assuming you found some enzyme combination that flanks the blue paper gene and also exists in the yellow paper circle, cut along the lines you've drawn and combine the DNA elements. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#*Note 1:  the yellow circle must include the small gray box since that symbolizes elements needed for the plasmid to survive, e.g. the replication origin and the antibiotic marker. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#*Note 2: the blue sequence must contain all of the gene you are trying to clone. If you choose an enzyme that cuts inside of the gene then you will be losing information that is needed for its expression. Choose a different pair of enzymes if you can. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===The main event: cloning pPRL or pGRN===</ins></div></td></tr>  </table>

Nkuldell http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=492641&oldid=prev Nkuldell at 10:50, 10 February 2011 2011-02-10T10:50:14Z

<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 10:50, 10 February 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 95:</td> <td colspan="2" class="diff-lineno">Line 95:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Peter Carr, USA (Custom).JPG|thumb|left|300px| Dr. Peter Carr]] Thanks for coming to talk to our class today.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Peter Carr, USA (Custom).JPG|thumb|left|300px| Dr. Peter Carr]] Thanks for coming to talk to our class today.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Plan B: U-do-it: DNA construction, recombinant DNA and PCR==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Plan B: U-do-it: DNA construction, recombinant DNA and PCR==</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Recombinant DNA technology relies heavily on genome construction tools. Restriction enzymes are at the heart of the molecular biology and biotechnology line-up. They are proteins that recognize a short sequence of DNA, bind to it and then clip the phosphate backbone of both the top and bottom strand. Today we will get some practice looking at restriction sites and how the sequences and enzymes can be used to clone a gene. At the end of the day we will set up an amplification reaction--another key, enabling technology for synthetic biology.  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">[[Image:200px-Mod1 1 eco ri.jpg| thumb| right| 300px| EcoR1 cut from 20.109, OCW]]</ins>Recombinant DNA technology relies heavily on genome construction tools. Restriction enzymes are at the heart of the molecular biology and biotechnology line-up. They are proteins that recognize a short sequence of DNA, bind to it and then clip the phosphate backbone of both the top and bottom strand. Today we will get some practice looking at restriction sites and how the sequences and enzymes can be used to clone a gene. At the end of the day we will set up an amplification reaction--another key, enabling technology for synthetic biology.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Warm up: Restriction enzymes to clone a paper gene===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Warm up: Restriction enzymes to clone a paper gene===</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>This exercise is adapted from <del class="diffchange diffchange-inline">[</del>[http://www.accessexcellence.org/RC/AB/WYW/wkbooks/SFTS/activity6.php access excellence.]<del class="diffchange diffchange-inline">]. </del>You will need a yellow, blue and white page ahowing DNA sequences and enzyme recognition sites.  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>This exercise is adapted from [http://www.accessexcellence.org/RC/AB/WYW/wkbooks/SFTS/activity6.php access excellence.] You will need a yellow, blue and white page ahowing DNA sequences and enzyme recognition sites.  </div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Begin by cutting the blue page into strips and taping the strips together according to the numbers at the bottom of each strip. In the end you will have a line (not a circle) of paper, with a grey shaded section within it. This grey section is the gene you'd like to clone but on the genomic DNA.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Begin by cutting the blue page into strips and taping the strips together according to the numbers at the bottom of each strip. In the end you will have a line (not a circle) of paper, with a grey shaded section within it. This grey section is the gene you'd like to clone but on the genomic DNA.</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Next scan this blue strip of paper for the enzyme restriction sites. You are welcome to look for all the enzymes, but in the interest of time today, please only identify on the paper where you can find the DNA recognition sequence for <del class="diffchange diffchange-inline"> </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Next scan this blue strip of paper for the enzyme restriction sites. You are welcome to look for all the enzymes, but in the interest of time today, please only identify on the paper where you can find the DNA recognition sequence for <ins class="diffchange diffchange-inline">'''Enzyme 3, Enzyme 6 and Enzyme 9.''' When you find them, you can mark on the paper</ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#</div></td></tr>  </table>

Nkuldell http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=492587&oldid=prev Nkuldell at 02:58, 10 February 2011 2011-02-10T02:58:58Z

<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 02:58, 10 February 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 94:</td> <td colspan="2" class="diff-lineno">Line 94:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==<font color = blue> Welcome Pete Carr!</font color>==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==<font color = blue> Welcome Pete Carr!</font color>==</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Peter Carr, USA (Custom).JPG|thumb|left|300px| Dr. Peter Carr]] Thanks for coming to talk to our class today.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Peter Carr, USA (Custom).JPG|thumb|left|300px| Dr. Peter Carr]] Thanks for coming to talk to our class today.</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;">==Plan B: U-do-it: DNA construction, recombinant DNA and PCR==</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Recombinant DNA technology relies heavily on genome construction tools. Restriction enzymes are at the heart of the molecular biology and biotechnology line-up. They are proteins that recognize a short sequence of DNA, bind to it and then clip the phosphate backbone of both the top and bottom strand. Today we will get some practice looking at restriction sites and how the sequences and enzymes can be used to clone a gene. At the end of the day we will set up an amplification reaction--another key, enabling technology for synthetic biology. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">===Warm up: Restriction enzymes to clone a paper gene===</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">This exercise is adapted from [[http://www.accessexcellence.org/RC/AB/WYW/wkbooks/SFTS/activity6.php access excellence.]]. You will need a yellow, blue and white page ahowing DNA sequences and enzyme recognition sites. </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"># Begin by cutting the blue page into strips and taping the strips together according to the numbers at the bottom of each strip. In the end you will have a line (not a circle) of paper, with a grey shaded section within it. This grey section is the gene you'd like to clone but on the genomic DNA.</ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Next scan this blue strip of paper for the enzyme restriction sites. You are welcome to look for all the enzymes, but in the interest of time today, please only identify on the paper where you can find the DNA recognition sequence for  </ins></div></td></tr> <tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#</ins></div></td></tr>  </table>

Nkuldell http://www.openwetware.org/index.php?title=2020%28S11%29_Lecture:week_2&diff=489327&oldid=prev Nkuldell at 15:41, 29 January 2011 2011-01-29T15:41:54Z

<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 15:41, 29 January 2011</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 88:</td> <td colspan="2" class="diff-lineno">Line 88:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div style="padding: .4em .9em .9em"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div style="padding: .4em .9em .9em"></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center>'''HOMEWORK'''</center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center>'''HOMEWORK'''</center></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Tomorrow we are lucky enough to have one of the premier genome engineers coming to speak with us. Pete Carr has done remarkable work in the MIT Media lab as part of the Center for Bits and Atoms, and has collaborated with George Church at Harvard Medical School on an ambitious genome rewriting project called "Re.coli." In advance of his talk tomorrow, please spend one hour looking at the following [[Media:GenomeEngineering Carr NatBiot09.pdf review article.]]  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Tomorrow we are lucky enough to have one of the premier genome engineers coming to speak with us. Pete Carr has done remarkable work in the MIT Media lab as part of the Center for Bits and Atoms, and has collaborated with George Church at Harvard Medical School on an ambitious genome rewriting project called "Re.coli." In advance of his talk tomorrow, please spend one hour looking at the following [[Media:GenomeEngineering Carr NatBiot09.pdf<ins class="diffchange diffchange-inline">| </ins>review article.]] <ins class="diffchange diffchange-inline"><br> Also, before Friday, please add some thoughts to the class [http://2020blog.21classes.com/home blog]. This will be the first of 6 entries you'll make this term.  </ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=<center>Week 2 Thursday</center>=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=<center>Week 2 Thursday</center>=</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==<font color = blue> Welcome Pete Carr!</font color>==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==<font color = blue> Welcome Pete Carr!</font color>==</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;">[[Image:Peter Carr, USA (Custom).JPG|thumb|left|300px| Dr. Peter Carr]] Thanks for coming to talk to our class today.</ins></div></td></tr>  </table>

Nkuldell