Team:Lethbridge/Lab Work

From 2010.igem.org

(Difference between revisions)

Latest revision as of 00:44, 18 October 2010

Here you can check out the work we have done in the lab, click on a month to take a look!

@@ Line 1: / Line 1: @@
-<b><font size=+2>April 13/2010</font> (In the Lab: JV, AS)</b><br>
+<div style="background-color:#000000; color:white">
-<b>Objective:</b> Test Restriction Endonucleases for Activity<br>
+<html>
-<b>Relevant Information:</b><br>
-Endonucleases available
+<br>
-<table><table border="3">
-<tr><td>Endonuclease</td><td>Optimal Buffer**</td><td>Other Buffers</td></tr>
+<table border="0" width="100%" style="background-color:#000000">
-<tr><td>EcoRV</td><td>None</td><td>2xT(100%); O,G(50-100%)</td></tr>
-<tr><td>EcoRI</td><td>Red</td><td>O(100%);R(100%)*;2xT(100%)</td></tr>
+<tr>
-<tr><td>BcuI/SpeI</td><td>Tango</td><td>B(50-100%);G(50-100%)</td></tr>
-<tr><td>XbaI</td><td>Tango</td><td>B,G,2xT(50-100%)</td></tr>
+<th>
-<tr><td>PstI</td><td>Orange</td><td>R(100%); B,G,T,2xT(50-100%)</td></tr>
-<tr><td>DpnI</td><td>Tango</td><td>B,G(100%): O,R,2xT(50-100%)</td></tr>
+<image src="https://static.igem.org/mediawiki/2010/2/29/UofLteamlogo.jpg" width="200px"/>
+</th>
+<th>
+<image src="https://static.igem.org/mediawiki/2010/9/91/UofLLabWork.JPG" height="300px"/>
+</th>
+<th>
+<image src="https://static.igem.org/mediawiki/2010/2/29/UofLteamlogo.jpg" width="200px"/>
+</th>
+</tr>
 </table>
-*Star Activity<br>
-**Optimal Buffer from Fermentas<br><br>
-Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV <br>
-<u>Red Buffer:</u> EcoRI, PstI, Control (No Enzyme)<br>
-<u>Tango Buffer:</u> BcuI/SpeI, XbaI, DpnI, Control (No Enzyme><br><br>
-<u>Methods:</u>
-Set up Master Mixes:
-<table><table border="3">
-<tr><td><b>Red MM</b></td><td>per tube (&micro;L)</td><td>Total (&micro;L)</td></tr>
-<tr><td>MilliQ H<sub>2</sub>0</td><td>13.75</td><td>55</td></tr>
-<tr><td>Red Buffer (10x)</td><td>2</td><td>7</td></tr>
-<tr><td>pUC19 (10pg/&micro;L)</td><td>2</td><td>7</td></tr>
-<tr><td><b>Total</b></td><td>19.75</td><td>69</td></tr>
-</table><br>
-<table><table border="3">
-<tr><td><b>Tango MM</b></td><td>per tube (&micro;L)</td><td>Total (&micro;L)</td></tr>
-<tr><td>MilliQ H<sub>2</sub>0</td><td>13.75</td><td>55</td></tr>
-<tr><td>Tango Buffer (10x)</td><td>2</td><td>7</td></tr>
-<tr><td>pUC19 (10pg/&micro;L)</td><td>2</td><td>7</td></tr>
-<tr><td><b>Total</b></td><td>19.75</td><td>69</td></tr>
-</table><br>
-To each tube, add <b>19.75&micro;L</b> of master mix and <b>0.25&micro;L</b> of enzyme<br>
-Incubated reaction mixes at 37<sup>o</sup>C (Start:7:00pm; End:7:45pm)<br>
-Add 3.3&micro;L of 6x loading dye to each reaction mixture and load 10&micro;L final volume onto a 1% agarose (in TAE) gel.<br>
-Add 1&micro;L of 6x loading dye to 1&micro;L of GeneRuler 1kb ladder (at 0.5&micro;g/&micro;L)<br>
-Gel loading order as follows:<br>
-<table><table border="3">
-<tr><td><b>Lane</b></td><td><b>Sample</b></td></tr>
-<tr><td>1</td><td>1kb Ladder</td></tr>
-<tr><td>2</td><td>Tango Control</td></tr>
-<tr><td>3</td><td>DpnI (Tango)</td></tr>
-<tr><td>4</td><td>BcuI/SpeI (Tango)</td></tr>
-<tr><td>5</td><td>XbaI (Tango)</td></tr>
-<tr><td>6</td><td>EcoRI (Red)</td></tr>
-<tr><td>7</td><td>PstI (Red)</td></tr>
-<tr><td>8</td><td>Red Control</td></tr>
-<tr><td>9</td><td>Empty</td></tr>
-<tr><td>10</td><td>Empty</td></tr>
-</table><br>
-Ran gel at 100V for 1 hour<br>
-<b>Results:</b> pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain). <br>
-<b>Conclusion:</b> Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining<br><br><br>
-<a name="may"></a>
-<b><font size=+2>May 5/2010</font>(in the lab: JV)</b><br>
+<br>
-<b>Objective:</b> Test Restriction Endonucleases for activity (take 2)<br>
-<b>Relevant Information:</b><br>
+<align="centre">
-Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks<br>
+<table border="0"  width="100%"  style="background-color:#000000">
-Prefix Enzymes are: EcoRI and XbaI<br>
-Suffix Enyzmes are: SpeI and PstI<br>
+<tr>
-(JV worked out in lab notebook which buffers would be best for each prefix/suffix enzyme combination)<br>
-Reactions will be assembled as follows:<br>
+<th>
-<table><table border="3">
-<tr><td><b>Enzyme</td><td>Buffer</td><td>Volume MM(&micro;L)</td><td>Volume Enzyme(&micro;L)</td></tr></b>
+<a href="https://2010.igem.org/Team:Lethbridge">
-<tr><td>PstI</td><td>Red</td><td>19.75</td><td>.25</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/2/22/UofLHome.jpg" width="80"/>
-<tr><td>XbaI</td><td>Tango</td><td>19.75</td><td>.25</td></tr>
+</a>
-<tr><td>SpeI</td><td>Tango</td><td>19.75</td><td>.25</td></tr>
-<tr><td>EcoRI</td><td>Red</td><td>19.75</td><td>.25</td></tr>
+</th>
-<tr><td>EcoRI/SpeI</td><td>Red</td><td>19.5</td><td>.25+.25</td></tr>
-<tr><td>XbaI/SpeI</td><td>Tango</td><td>19.5</td><td>.25+.25</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Team">
-<tr><td>EcoRI/PstI</td><td>Red</td><td>19.5</td><td>.25+.25</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/0/0d/UofLTeam.jpg" width="80"/>
-<tr><td>XbaI/PstI</td><td>Tango</td><td>19.5</td><td>.25+.25</td></tr>
+</a>
-</table><br>
+</th>
-Make up Master Mixes as follows:<br>
-<table><table border="3">
+<th><a href="https://2010.igem.org/Team:Lethbridge/Project">
-<tr><td><b>Red MM</td><td>per tube(&micro;L)</td><td>Total*(&micro;L)</td></tr></b>
+<img src="https://static.igem.org/mediawiki/2010/8/8d/UofLProjectbutton.jpg" width="80"/>
-<tr><td>MilliQ H<sub>2</sub>0</td><td>15.75</td><td>86.675</td></tr>
+</a>
-<tr><td>Red Buffer (10x)</td><td>2</td><td>11</td></tr>
+</th>
-<tr><td>pDNA**</td><td>2</td><td>11</td></tr>
-</table><br>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work">
-<table><table border="3">
+<img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/>
-<tr><td><b>Tango MM</td><td>per tube(&micro;L)</td><td>Total*(&micro;L)</td></tr></b>
+</a>
-<tr><td>MilliQ H<sub>2</sub>0</td><td>15.75</td><td>86.675</td></tr>
+</th>
-<tr><td>Tango Buffer (10x)</td><td>2</td><td>11</td></tr>
-<tr><td>pDNA**</td><td>2</td><td>11</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Parts">
+<img src="https://static.igem.org/mediawiki/2010/8/84/UofLPartsSubmittedToTheRegistrybutton.jpg" width="80"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Modeling">
+<img src="https://static.igem.org/mediawiki/2010/e/e1/UofLModelingbutton.jpg" width="80"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics">
+<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Safety">
+<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Art">
+<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/News">
+<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/>
+</a>
+</th>
 </table>
-*Volume per reaction multiplied by 5.5<br>
+</body>
-**Unknown concentration of pDNA<br><br>
+</html>
-Incubated for 70min at 37<sup>o</sup>C (Start-1:05pm; End-2:15pm)<br>
+<hr>
-Added 3.3&micro;L of 6x loading dye to each reaction mixture and loaded 10&micro;L onto a 1% agarose gel (in TAE)<br>
-Added 1&micro;L of 6x loading dye to 2&micro;L of gene ruler 1kb ladder<br>
-Load order as follows:<br>
-<table><table border="3">
-<tr><td><b>Lane</td><td>Sample</td><td>Volume Loaded (&micro;L)</td></tr></b>
-<tr><td>1</td><td>pSB-CEYFP/PstI</td><td>10</td></tr>
-<tr><td>2</td><td>pSB-CEYFP/EcoRI</td><td>10</td></tr>
-<tr><td>3</td><td>pSB-CEYFP/EcoRI/PstI</td><td>10</td></tr>
-<tr><td>4</td><td>pSB-CEYFP/EcoRI/SpeI</td><td>10</td></tr>
-<tr><td>5</td><td>pSB-CEYFP/XbaI/PstI</td><td>10</td></tr>
-<tr><td>6</td><td>pSB-CEYFP/XbaI</td><td>10</td></tr>
-<tr><td>7</td><td>pSB-CEYFP/SpeI</td><td>10</td></tr>
-<tr><td>8</td><td>pSB-CEYFP/XbaI/SpeI</td><td>10</td></tr>
-<tr><td>9</td><td>pSB-CEYFP/Red Master Mix Control</td><td>10</td></tr>
-<tr><td>10</td><td>pSB-CEYFP/Tango Master Mix Control</td><td>10</td></tr>
-<tr><td>11</td><td>pSB-CEYFP/MilliQ H<sub>2</sub>0 Control</td><td>10</td></tr>
-<tr><td>12</td><td>Ladder</td><td>4</td></tr>
-</table><br>
-Ran gel at 100V for 1 hour<br><br>
-<b>Results:</b><br>
-[[image:100505JV-EnzymeTest1Cropped.jpg|200px|none]]
-This gel shows that SpeI does not cut on its own, and does not cut when combined with other enzymes<br>
-<b>Conclusion:</b> Test other source of SpeI to see if it has any activity.<br><br>
-<b><font size=+2>May 6/2010</font>(in the lab:KG, AS)</b><br>
+<html>
-<b>Objective:</b> To check if the old SpeI enzyme (exp date: March 2011) will cleave plasmid DNA, since we believe the newer SpeI enzyme (exp date: 2012) does not.<br>
+<body>
-<b>Method:</b><br>
+<center>
-<table><table border ="3">
+<table border="0"  width="28%"  style="background-color:#000000">
-<tr><td><b>Red Master Mix</b></td><td>per tube (&micro;L)</td><td>Total Volume*</td></tr>
-<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75</td><td>63</td></tr>
+<tr>
-<tr><td>Red Buffer (10x)</td><td>2</td><td>8</td></tr>
+<th>
-<tr><td>pDNA**</td><td>2</td><td>8</td></tr>
+<div class="miniBar">
-</table>
+		<div class="countdown"><object type="application/x-shockwave-flash" data="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=10&date_day=27&date_year=0&un=THE WIKI FREEZE&size=normal&mo=10&da=27&yr=2010" width="300" height="100"><param name="movie" value="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=10&date_day=27&date_year=0&un=THE WIKI FREEZE&size=normal&mo=10&da=27&yr=2010" /><param name="bgcolor" value="#000000" /></object><img src="http://www.oneplusyou.com/q/img/bb_badges/countdown.jpg" alt="" style="display: none;" height="1" width="1" /></div>
-*Volume per tube multiplied by 4<br>
+		<div class="miniContainer">
-**Used pSB NEYFP pDNA from cell E5 in plasmid box<br>
-Enzymes that will use Red Master Mix are: EcoRI+SpeI (old), EcoRI+SpeI (new)<br>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work">
-Add 0.25&micro;L of each enzyme to 19.5&micro;L of master mix<br><br>
+<img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/>
-<table><table border ="3">
+</a>
-<tr><td><b>Tango Master Mix</b></td><td>per tube (&micro;L)</td><td>Total Volume*</td></tr>
+</th>
-<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75</td><td>94.5</td></tr>
-<tr><td>Tango Buffer (10x)</td><td>2</td><td>12</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols">
-<tr><td>pDNA**</td><td>2</td><td>12</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/9/91/UofLprotocolsbutton.jpg" width="60"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Calendar">
+<img src="https://static.igem.org/mediawiki/2010/7/73/UofLcalendar.jpg" width="60"/>
+</a>
+</th>
+<th>
+<div class="miniBar">
+		<div class="countdown"><object type="application/x-shockwave-flash" data="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=11&date_day=05&date_year=0&un=THE IGEM JAMBOREE&size=normal&mo=11&da=05&yr=2010" width="300" height="100"><param name="movie" value="http://www.oneplusyou.com/bb/files/countdown/countdown.swf?co=FFFFFF&bgcolor=000000&date_month=11&date_day=05&date_year=0&un=THE IGEM JAMBOREE&size=normal&mo=11&da=05&yr=2010" /><param name="bgcolor" value="#000000" /></object><img src="http://www.oneplusyou.com/q/img/bb_badges/countdown.jpg" alt="" style="display: none;" height="1" width="1" /></div>
+		<div class="miniContainer">
+</th>
+<tr>
 </table>
-*Volume per tube multiplied by 6<br>
+</center>
-**Used pSB NEYFP pDNA from cell E5 in plasmid box<br>
+</body>
-Enzymes that will use Tango Master Mix are: SpeI (old), SpeI (new), XbaI+SpeI (old), XbaI+SpeI (new)<br>
+</html>
-Add 0.25&micro;L of each enzyme to 19.5&micro;L of master mix<br><br>
-Incubated all reactions at 37<sup>o</sup>C for 1h (Start-8:30pm; End-9:30pm)<br>
-Will not be able to run on agarose gel tonight, will label them so JV can run them in the morning<br>
-<u>Tube Names:</u><br>
-Master Mix 1 Control (Red Buffer)<br>
-Master Mix 2 Control (Tango Buffer)<br>
-E+S(N); EcoRI + SpeI(N)<br>
-E+S(O); EcoRI + SpeI(O)<br>
-X+S(N); XbaI + SpeI(N)<br>
-X+S(O); XbaI + SpeI(O)<br>
-S(N); SpeI(N)<br>
-S(O); SpeI(O)<br><br>
-Placed in -20<sup>o</sup>C freezer of later analysis by agarose electrophoresis<br><br>
+<hr>
-<b><font size=+2>May 10/2010</font>(in the lab:JV)</b><br>
+<html>
-<b>Objective:</b> To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis<br><br>
+<center>
-<b>Method:</b><br>
+<font color="white">Here you can check out the work we have done in the lab, click on a month to take a look!
-<table><table border="3">
+</center>
-<tr><td><b>Lane</b></td><td>Sample</td><td>Quantity Loaded (&micro;L)</td></tr>
+</html>
-<tr><td>1</td><td>MM1 Control</td><td>10</td></tr>
-<tr><td>2</td><td>MM2 Control</td><td>10</td></tr>
+<html>
-<tr><td>3</td><td>EcoRI+SpeI(N)</td><td>10</td></tr>
+<body>
-<tr><td>4</td><td>EcoRI+SpeI(O)</td><td>10</td></tr>
+<center>
-<tr><td>5</td><td>SpeI(N)</td><td>10</td></tr>
+<table border="0"  width="50%"  style="background-color:#000000">
-<tr><td>6</td><td>SpeI(O)</td><td>10</td></tr>
-<tr><td>7</td><td>XbaI+SpeI(N)</td><td>10</td></tr>
+<tr>
-<tr><td>8</td><td>XbaI+SpeI(O)</td><td>10</td></tr>
-<tr><td>9</td><td>1kb Ladder</td><td>5</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/April">
+<img src="https://static.igem.org/mediawiki/2010/8/8a/UofLapril.jpg" width="60"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/May">
+<img src="https://static.igem.org/mediawiki/2010/7/7b/UofLmaybutton.jpg" width="60"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/June">
+<img src="https://static.igem.org/mediawiki/2010/8/80/UofLjunebutton.jpg" width="60"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/July">
+<img src="https://static.igem.org/mediawiki/2010/5/53/UofLjulybutton.jpg" width="60"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/August">
+<img src="https://static.igem.org/mediawiki/2010/1/15/UofLaugustbutton.jpg" width="60"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/September">
+<img src="https://static.igem.org/mediawiki/2010/4/4d/UofLseptemberbutton.jpg" width="60"/>
+</a>
+</th>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/October">
+<img src="https://static.igem.org/mediawiki/2010/4/4e/UofLoctoberbutton.jpg" width="60"/>
+</a>
+</th>
+<tr>
 </table>
-Run gel for 60min at 100V<br><br>
+</center>
-<b>Results:</b><br>
+</body>
-[[image:100510JRV-EnzymeTest1Cropped.jpg|200px|none]]<br>
+</html>
-It appears as though both SpeI enzymes are working properly here. We will utilize the newer batch of SpeI (expires 2012) from this point forward.<br><br>
+<hr>
+<BLOCKQUOTE>
+<br>