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- | <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> | | </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 (µL)</td><td>Total (µL)</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/µL)</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 (µL)</td><td>Total (µL)</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/µL)</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>19.75µL</b> of master mix and <b>0.25µL</b> of enzyme<br>
| |
- | Incubated reaction mixes at 37<sup>o</sup>C (Start:7:00pm; End:7:45pm)<br>
| |
- | Add 3.3µL of 6x loading dye to each reaction mixture and load 10µL final volume onto a 1% agarose (in TAE) gel.<br>
| |
- | Add 1µL of 6x loading dye to 1µL of GeneRuler 1kb ladder (at 0.5µg/µL)<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(µL)</td><td>Volume Enzyme(µL)</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(µL)</td><td>Total*(µL)</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(µL)</td><td>Total*(µL)</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> | | </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 3.3µL of 6x loading dye to each reaction mixture and loaded 10µL onto a 1% agarose gel (in TAE)<br>
| + | |
- | Added 1µL of 6x loading dye to 2µL 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 (µL)</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 (µL)</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 0.25µL of each enzyme to 19.5µL 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 (µL)</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> | | </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 0.25µL of each enzyme to 19.5µL 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 (µL)</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> | | </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> |