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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
| + | |
| - | <table><table border="3">
| + | |
| - | <tr><td>Endonuclease</td><td>Optimal Buffer**</td><td>Other Buffers</td></tr>
| + | |
| - | <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><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>
| + | |
| - | <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>
| + | |
| - | </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>
| + | |
| | | | |
| | + | <br> |
| | | | |
| - | <a name="may"></a> | + | <table border="0" width="100%" style="background-color:#000000"> |
| - | <b><font size=+2>May 5/2010</font>(in the lab: JV)</b><br>
| + | |
| - | <b>Objective:</b> Test Restriction Endonucleases for activity (take 2)<br>
| + | <tr> |
| - | <b>Relevant Information:</b><br>
| + | |
| - | Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks<br>
| + | <th> |
| - | Prefix Enzymes are: EcoRI and XbaI<br>
| + | |
| - | Suffix Enyzmes are: SpeI and PstI<br>
| + | <image src="https://static.igem.org/mediawiki/2010/2/29/UofLteamlogo.jpg" width="200px"/> |
| - | (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>
| + | <th> |
| - | <tr><td>PstI</td><td>Red</td><td>19.75</td><td>.25</td></tr>
| + | |
| - | <tr><td>XbaI</td><td>Tango</td><td>19.75</td><td>.25</td></tr>
| + | <image src="https://static.igem.org/mediawiki/2010/9/91/UofLLabWork.JPG" height="300px"/> |
| - | <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> |
| - | <tr><td>EcoRI/PstI</td><td>Red</td><td>19.5</td><td>.25+.25</td></tr> | + | |
| - | <tr><td>XbaI/PstI</td><td>Tango</td><td>19.5</td><td>.25+.25</td></tr>
| + | <image src="https://static.igem.org/mediawiki/2010/2/29/UofLteamlogo.jpg" width="200px"/> |
| - | </table><br>
| + | |
| - | Make up Master Mixes as follows:<br>
| + | </th> |
| - | <table><table border="3">
| + | |
| - | <tr><td><b>Red MM</td><td>per tube(µL)</td><td>Total*(µL)</td></tr></b>
| + | </tr> |
| - | <tr><td>MilliQ H<sub>2</sub>0</td><td>15.75</td><td>86.675</td></tr>
| + | |
| - | <tr><td>Red Buffer (10x)</td><td>2</td><td>11</td></tr>
| + | |
| - | <tr><td>pDNA**</td><td>2</td><td>11</td></tr>
| + | |
| - | </table><br> | + | |
| - | <table><table border="3"> | + | |
| - | <tr><td><b>Tango MM</td><td>per tube(µL)</td><td>Total*(µL)</td></tr></b>
| + | |
| - | <tr><td>MilliQ H<sub>2</sub>0</td><td>15.75</td><td>86.675</td></tr>
| + | |
| - | <tr><td>Tango Buffer (10x)</td><td>2</td><td>11</td></tr>
| + | |
| - | <tr><td>pDNA**</td><td>2</td><td>11</td></tr>
| + | |
| - | </table>
| + | |
| - | *Volume per reaction multiplied by 5.5<br>
| + | |
| - | **Unknown concentration of pDNA<br><br>
| + | |
| - | Incubated for 70min at 37<sup>o</sup>C (Start-1:05pm; End-2:15pm)<br>
| + | |
| - | 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>
| |
| - | <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>
| |
| - | <b>Method:</b><br>
| |
| - | <table><table border ="3">
| |
| - | <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><td>Red Buffer (10x)</td><td>2</td><td>8</td></tr>
| |
| - | <tr><td>pDNA**</td><td>2</td><td>8</td></tr>
| |
| - | </table>
| |
| - | *Volume per tube multiplied by 4<br>
| |
| - | **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>
| |
| - | Add 0.25µL of each enzyme to 19.5µL of master mix<br><br>
| |
| - | <table><table border ="3">
| |
| - | <tr><td><b>Tango 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>94.5</td></tr>
| |
| - | <tr><td>Tango Buffer (10x)</td><td>2</td><td>12</td></tr>
| |
| - | <tr><td>pDNA**</td><td>2</td><td>12</td></tr>
| |
| | </table> | | </table> |
| - | *Volume per tube multiplied by 6<br>
| |
| - | **Used pSB NEYFP pDNA from cell E5 in plasmid box<br>
| |
| - | Enzymes that will use Tango Master Mix are: SpeI (old), SpeI (new), XbaI+SpeI (old), XbaI+SpeI (new)<br>
| |
| - | 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>
| |
| | | | |
| | | | |
| - | <b><font size=+2>May 10/2010</font>(in the lab:JV)</b><br> | + | |
| - | <b>Objective:</b> To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis<br><br> | + | <br> |
| - | <b>Method:</b><br> | + | |
| - | <table><table border="3">
| + | <align="centre"> |
| - | <tr><td><b>Lane</b></td><td>Sample</td><td>Quantity Loaded (µL)</td></tr>
| + | <table border="0" width="100%" style="background-color:#000000"> |
| - | <tr><td>1</td><td>MM1 Control</td><td>10</td></tr> | + | |
| - | <tr><td>2</td><td>MM2 Control</td><td>10</td></tr> | + | <tr> |
| - | <tr><td>3</td><td>EcoRI+SpeI(N)</td><td>10</td></tr> | + | |
| - | <tr><td>4</td><td>EcoRI+SpeI(O)</td><td>10</td></tr> | + | <th> |
| - | <tr><td>5</td><td>SpeI(N)</td><td>10</td></tr> | + | |
| - | <tr><td>6</td><td>SpeI(O)</td><td>10</td></tr> | + | <a href="https://2010.igem.org/Team:Lethbridge"> |
| - | <tr><td>7</td><td>XbaI+SpeI(N)</td><td>10</td></tr> | + | <img src="https://static.igem.org/mediawiki/2010/2/22/UofLHome.jpg" width="80"/> |
| - | <tr><td>8</td><td>XbaI+SpeI(O)</td><td>10</td></tr> | + | </a> |
| - | <tr><td>9</td><td>1kb Ladder</td><td>5</td></tr> | + | |
| | + | </th> |
| | + | |
| | + | <th><a href="https://2010.igem.org/Team:Lethbridge/Team"> |
| | + | <img src="https://static.igem.org/mediawiki/2010/0/0d/UofLTeam.jpg" width="80"/> |
| | + | </a> |
| | + | </th> |
| | + | |
| | + | <th><a href="https://2010.igem.org/Team:Lethbridge/Project"> |
| | + | <img src="https://static.igem.org/mediawiki/2010/8/8d/UofLProjectbutton.jpg" width="80"/> |
| | + | </a> |
| | + | </th> |
| | + | |
| | + | <th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work"> |
| | + | <img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/> |
| | + | </a> |
| | + | </th> |
| | + | |
| | + | <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> |
| - | Run gel for 60min at 100V<br><br>
| + | </body> |
| - | <b>Results:</b><br>
| + | </html> |
| - | [[image:100510JRV-EnzymeTest1Cropped.jpg|200px|none]]<br>
| + | <hr> |
| - | 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>
| + | |
| | | | |
| - | <b><font size=+2>May 10/2010</font>(in the lab:JV, KG, AV)</b><br> | + | <html> |
| - | <b>Objective:</b>Make 24 LB agar plates with 100µg/mL ampicillin antibiotic.<br> | + | <body> |
| - | <b>Method:</b>Make 2L of LB media with agar<br> | + | <center> |
| - | 2x10g Tryptone<br>
| + | <table border="0" width="28%" style="background-color:#000000"> |
| - | 2X2.5g Yeast Extract<br>
| + | |
| - | 2x5g NaCl<br>
| + | |
| - | 2x10g Agar<br><br>
| + | |
| - | Continued <b> May 11/2010<br></b>
| + | |
| - | (Stock Ampicillin solution is 100mg/mL)<br>
| + | |
| - | Have 4x500mL of LB with Agar<br>
| + | |
| - | Add 500µL of stock ampicillin to 500mL of media<br><br>
| + | |
| - | <b><font size=+2>May 11/2010 Evening </font> (in the lab: KG, AV, MC, TF, JV, JS)<br></b>
| + | |
| - | <b>Objective:</b> To transform the following plasmids into DH5α <i>E.coli</i> cells.
| + | |
| - | <table><table border=" 3" > | + | |
| - | <tr><td><b>Construct Name (2009)</td></b><td><b>Construct Location (2009)</b></td></tr>
| + | |
| - | <tr><td>Lumazine</td><td>J4</td></tr>
| + | |
| - | <tr><td>Lumazine-dT</td><td>J5,J6</td></tr>
| + | |
| - | <tr><td>sRBS-Lumazine-dT</td><td>J7,J8</td></tr>
| + | |
| - | <tr><td>pBAD-TetR</td><td>I4</td></tr>
| + | |
| - | <tr><td>pBAD</td><td>A5,F10</td></tr>
| + | |
| - | <tr><td>sRBS</td><td>D5,E10</td></tr>
| + | |
| - | <tr><td>pSB-CEYFP</td><td>E5,D6</td></tr>
| + | |
| - | <tr><td>pSB-NEYFP</td><td>F5,C6</td></tr>
| + | |
| - | <tr><td>C-term Tag</td><td>C10</td></tr>
| + | |
| - | <tr><td>N-term Tag</td><td>D9,D10</td></tr>
| + | |
| - | <tr><td>pTet</td><td>E4</td></tr>
| + | |
| - | <tr><td>EYFP</td><td>A4</td></tr>
| + | |
| - | <tr><td>CFP Complete</td><td>D4</td></tr>
| + | |
| - | </table><br>
| + | |
| - | <b>Method: </b>Followed [https://2010.igem.org/Team:Lethbridge/Notebook/Protocol Competent Cell Transformation] protocol in Common Protocols section and plated on LB agar supplemented with ampicillin.<br>
| + | |
| - | <b>Results: </b>The following plasmids were successfully transformed and formed colonies:<br>
| + | |
| - | <ul>
| + | |
| - | <li>Lumazine (J4)</li>
| + | |
| - | <li>sRBS-Lumazine-dT (J7)</li>
| + | |
| - | <li>sRBS-Lumazine-dT (J8)</li>
| + | |
| - | <li>pBAD (A5) </li>
| + | |
| - | <li>pBAD (F10) </li>
| + | |
| - | <li>pSB-CEYFP </li>
| + | |
| - | <li>pSB-NEYFP </li>
| + | |
| - | <li>N-term tag </li>
| + | |
| - | <li>EYFP (A4)</li>
| + | |
| - | <li>CFP Complete (D4)</li></ul><br>
| + | |
| - | <b>Conclusion:</b> Need another attempt to transform the following plasmids:<br>
| + | |
| - | <ul>
| + | |
| - | <li>Lumazine-dT (J5,J6)</li>
| + | |
| - | <li>pBAD-TetR </li>
| + | |
| - | <li>sRBS (D5,E10)</li>
| + | |
| - | <li>C-Term tag</li>
| + | |
| - | <li>pTet</li></ul><br>
| + | |
| | | | |
| - | <br><b><font size=+2>May 12/2010</font>(in the lab: JV)</b><br> | + | <tr> |
| - | <b>Objective: Miniprep of plasmid DNA from transformed cells</b>(JV, AV, HB)<br> | + | <th> |
| - | <b>Method:</b><br>
| + | <div class="miniBar"> |
| - | <ul><li>Inoculate 5mL of LB liquid media (with 100µL/mL Ampicillin) with cells from competent cells plates (picked with sterile toothpick).</li>
| + | <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> |
| - | <li>Allow cells in liquid culture to grow overnight in 37<sup>o</sup>C shaking incubator (300RPM)
| + | <div class="miniContainer"> |
| - | Purify plasmid DNA from cells by using "Boiling Lysis Plasmid Preparation" protocol in Common Protocols Section.</li>
| + | |
| - | <li>CHANGE: Step 14, used MilliQ H<sub>2</sub>O (with 20ng/µL RNase A) instead of TE buffer.</li></ul><br> | + | <th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work"> |
| - | Plasmids were transferred to the "iGEM 2010 - Working Plasmid DNA" box in the -20<sup>o</sup>C freezer in the iGEM lab. Plasmids were placed in the following cells:
| + | <img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/> |
| - | <table><table border="3"> | + | </a> |
| - | <tr><td>Construct</td><td>Cell in Working Plasmid Box (2010)</td><td>Original Cell in Old Box</td></tr>
| + | </th> |
| - | <tr><td>sRBS-Lumazine-dT</td><td>A1</td><td>J7</td></tr>
| + | |
| - | <tr><td>sRNS-Lumazine-dT</td><td>A2</td><td>J8</td></tr>
| + | <th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols"> |
| - | <tr><td>CFP Complete</td><td>B6</td><td>D4</td></tr> | + | <img src="https://static.igem.org/mediawiki/2010/9/91/UofLprotocolsbutton.jpg" width="60"/> |
| - | <tr><td>Lumazine</td><td>A3</td><td>J4</td></tr> | + | </a> |
| - | <tr><td>pBAD</td><td>A4</td><td>A5</td></tr>
| + | </th> |
| - | <tr><td>pBAD</td><td>A5</td><td>F10</td></tr>
| + | |
| - | <tr><td>pSB-CEYFP</td><td>B5</td><td></td></tr> | + | <th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Calendar"> |
| - | <tr><td>pSB-NEYFP</td><td>B4</td><td></td></tr>
| + | <img src="https://static.igem.org/mediawiki/2010/7/73/UofLcalendar.jpg" width="60"/> |
| - | <tr><td>EYFP</td><td>B1</td><td>A4</td></tr> | + | </a> |
| - | <tr><td>N-term tag</td><td>B2</td><td></td></tr>
| + | </th> |
| - | </table><br> | + | |
| - | Also generated sterile glycerol stocks and placed in -80<sup>o</sup>C freezer in the 2010 iGEM box as follows:
| + | <th> |
| - | <table><table border="3"> | + | <div class="miniBar"> |
| - | <tr><td>Construct</td><td>Cell Working Glycerol Stock Box (2010)</td></tr>
| + | <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> |
| - | <tr><td>sRBS-Lumazine-dT (J7)</td><td>B2,C4,D2</td></tr>
| + | <div class="miniContainer"> |
| - | <tr><td>sRNS-Lumazine-dT (J8)</td><td>C6</td></tr> | + | </th> |
| - | <tr><td>CFP Complete</td><td>A10, C8</td></tr>
| + | <tr> |
| - | <tr><td>Lumazine</td><td>A8,B10</td></tr>
| + | |
| - | <tr><td>pBAD (from A5)</td><td>B5,B9</td></tr>
| + | |
| - | <tr><td>pBAD (from F10)</td><td>B3,B7</td></tr>
| + | |
| - | <tr><td>pSB-CEYFP</td><td>C3,B5</td></tr> | + | |
| - | <tr><td>pSB-NEYFP</td><td>B6,C1</td></tr> | + | |
| - | <tr><td>EYFP</td><td>C7,B8</td></tr> | + | |
| - | <tr><td>N-term tag</td><td>C2,D4</td></tr>
| + | |
| - | </table><br>
| + | |
| - | <b>Objective:</b> Restrict plasmid DNA with restriction endonucleases (JV)<br>
| + | |
| - | <b>Method:</b><br>
| + | |
| - | Have:
| + | |
| - | 10 lanes of restricted plasmid DNA <br>
| + | |
| - | 10 lanes of unrestricted plasmid DNA <br>
| + | |
| - | 1 lane of buffer control <br>
| + | |
| - | Use EcoRI (prefix cutter) and PstI (suffix cutter) <br><br>
| + | |
| - | Pipetting Scheme for Restriction Tubes:
| + | |
| - | <table><table border="3">
| + | |
| - | <tr><td>Ingredient</td><td>Volume/tube (µL)</td><td>Total Volume*</td></tr>
| + | |
| - | <tr><td>MilliQ H<sub>2</sub>O</td><td>15.5</td><td>155</td></tr>
| + | |
| - | <tr><td>Red Buffer (10X)</td><td>2</td><td>20</td></tr>
| + | |
| - | <tr><td>EcoRI</td><td>0.25</td><td>2.5</td></tr>
| + | |
| - | <tr><td>PstI</td><td>0.25</td><td>2.5</td></tr> | + | |
| | </table> | | </table> |
| - | *Amount per tube multiplied by 10<br>
| + | </center> |
| - | Pipetting Scheme for Unrestricted reactions:
| + | </body> |
| - | <table><table border="3">
| + | </html> |
| - | <tr><td><b>Ingredient</b></td><td>Volume/tube (µL)</td><td>Total Volume*</td></tr>
| + | |
| - | <tr><td>MilliQ H<sub>2</sub>O</td><td>16</td><td>160</td></tr>
| + | |
| - | <tr><td>Red Buffer (10X)</td><td>2</td><td>20</td></tr>
| + | |
| - | </table>
| + | |
| - | *Amount per tube multiplied by 10<br>
| + | |
| - | Buffer Control will be 18µL MilliQ H<sub>2</sub>O + 2µL 10x Red Buffer.<br>
| + | |
| - | Place in 37<sup>o</sup>C water bath at 2:55pm and removed at 4:57pm for a 2 hour incubation.<br>
| + | |
| - | Analyzed restriction digests on a 1% agarose gel (large gel apparatus ~70mL)<br>
| + | |
| - | Added 1µL of 6x DNA loading dye to 5µL of sample<br>
| + | |
| - | Added 2µL of 6x DNA loading dye to 6µL of TAE buffer and 2µL of 1kb DNA mass ladder.<br>
| + | |
| - | Loaded samples as follows:<br>
| + | |
| - | <table><table border="3">
| + | |
| - | <tr><td><b>Lane</td><td>Sample</td><td>Volume Loaded (µL)</b></td></tr>
| + | |
| - | <tr><td>1</td><td>1 kb Ladder</td><td>5</td></tr>
| + | |
| - | <tr><td>2</td><td>Buffer Control</td><td>5</td></tr>
| + | |
| - | <tr><td>3</td><td>pSB-NEYFP</td><td>5</td></tr>
| + | |
| - | <tr><td>4</td><td>Restricted Lumazine</td><td>5</td></tr>
| + | |
| - | <tr><td>5</td><td>Lumazine</td><td>5</td></tr>
| + | |
| - | <tr><td>6</td><td>Restricted pSB-NEYFP</td><td>5</td></tr>
| + | |
| - | <tr><td>7</td><td>pSB-CEYFP</td><td>5</td></tr>
| + | |
| - | <tr><td>8</td><td>Restricted pSB-CEYFP</td><td>5</td></tr>
| + | |
| - | <tr><td>9</td><td>pBAD</td><td>5</td></tr>
| + | |
| - | <tr><td>10</td><td>Restricted pBAD</td><td>5</td></tr>
| + | |
| - | <tr><td>11</td><td>EYFP</td><td>5</td></tr>
| + | |
| - | <tr><td>12</td><td>Restricted EYFP</td><td>5</td></tr>
| + | |
| - | <tr><td>13</td><td>CFP Complete</td><td>5</td></tr>
| + | |
| - | <tr><td>14</td><td>Restricted CFP Complete</td><td>5</td></tr>
| + | |
| - | <tr><td>15</td><td>sRBS-Lumazine-dT (J7)</td><td>5</td></tr>
| + | |
| - | <tr><td>16</td><td>Restricted sRBS-Lumazine-dT (J7)</td><td>5</td></tr>
| + | |
| - | <tr><td>17</td><td>N-term Tag</td><td>5</td></tr>
| + | |
| - | <tr><td>18</td><td>Restricted N-term Tag</td><td>5</td></tr>
| + | |
| - | <tr><td>19</td><td>sRBS-Lumazine-dT (J8)</td><td>5</td></tr>
| + | |
| - | <tr><td>20</td><td>Restricted sRBS-Lumazine-dT (J8)</td><td>5</td></tr>
| + | |
| - | </table><br>
| + | |
| - | Ran gel at 100V for 90 minutes (Start-9:50pm; End-11:20pm)<br>
| + | |
| - | Stained with ethidium bromide for 20 minutes.<br>
| + | |
| | | | |
| - | <b>Results:</b><br> | + | <hr> |
| - | [[image:100513TF.KG.JS-Plasmids_for_Seq.Cropped.jpg|200px|none]]
| + | |
| - | <b><font size=+2>May 13/2010 Evening</font>(in lab: AS,TF,KG,JS,MC)</b><br>
| + | |
| - | <b>Objective:</b> To make a second attempt at transforming plasmids that didn't transform the first time. These plasmids are:<br>
| + | |
| - | <ul>
| + | |
| - | <li>Lumazine-dT (J5,J6)</li>
| + | |
| - | <li>pBad-TetR</li>
| + | |
| - | <li>sRBS (D5,E10)</li>
| + | |
| - | <li>C-term tag</li>
| + | |
| - | <li>pTet</li></ul><br>
| + | |
| - | All DH5α cells were used up in the last transformation, had to aliquot an additional 50x 20µL aliquots (MC,TF)<br>
| + | |
| - | Transform plasmid DNA (Using "Competent Cell Transformation" Protocol) into newly aliquotted DH5α cells. (KG,JS)<br>
| + | |
| - | NOTES:<br>
| + | |
| - | AS concerned that there is something not quite right with LB liquid media added to transformed cells, but continued anyways (JV informed AS the next day that the LB liquid media had not been sterilized).<br>
| + | |
| - | Plated all 250µL of culture.<br>
| + | |
| - | <b>Results:</b><br>
| + | |
| - | <table><table border="3">
| + | |
| - | <tr><td><b>Construct</td><td>Result</b></td></tr>
| + | |
| - | <tr><td>Lumazine-dT(1)</td><td>Growth present</td></tr>
| + | |
| - | <tr><td>sRBS-Lumazine-dT</td><td>Growth present</td></tr>
| + | |
| - | <tr><td>sRBS (D5)</td><td>Growth present</td></tr>
| + | |
| - | <tr><td>sRBS (E10)</td><td>Growth present</td></tr>
| + | |
| - | <tr><td>C-term tag</td><td>No growth present</td></tr>
| + | |
| - | <tr><td>pTet</td><td>No growth present</td></tr>
| + | |
| - | </table><br>
| + | |
| - | <b>Next Steps:</b><br>
| + | |
| - | Make another attempt to transform the C-term tag and pTet constructs.<br>
| + | |
| - | Start overnight cultures of cells that grew for plasmid prep and sequencing.<br><br>
| + | |
| | | | |
| - | <b><font size=+2>May 14/2010</font>(in the lab: JV)</b><br> | + | <html> |
| - | <b>Objective:</b> Quantify pDNA concentration in order to ensure sufficient material for sequence analysis.<br>
| + | <center> |
| - | <b>Method:</b> Measure absorbance of samples at 260nm.<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! |
| - | <b>Results:</b><br>
| + | </center> |
| - | <table><table border="3">
| + | </html> |
| - | <tr><td><b>Sample</td><td>Absorbance at 260nm</b></td></tr>
| + | |
| - | <tr><td>sRBS-Lumazine-dT (J7)</td><td>0.311</td></tr>
| + | |
| - | <tr><td>sRBS-Lumazine-dT (J8)</td><td>0.309</td></tr>
| + | |
| - | <tr><td>CFP complete</td><td>0.316</td></tr>
| + | |
| - | <tr><td>N-term tag</td><td>0.290</td></tr>
| + | |
| - | <tr><td>pSB-CEYFP</td><td>0.338</td></tr>
| + | |
| - | <tr><td>pSB-NEYFP</td><td>0.403</td></tr>
| + | |
| - | <tr><td>pBAD (A5)</td><td>0.282</td></tr>
| + | |
| - | <tr><td>pBAD (F10)</td><td>0.562</td></tr>
| + | |
| - | <tr><td>EYFP</td><td>0.389</td></tr>
| + | |
| - | <tr><td>Lumazine</td><td>0.221</td></tr>
| + | |
| - | </table><br>
| + | |
| - | <b>Conclusion:</b> All plasmids present in sufficient concentrations for sequence analysis.<br>
| + | |
| | | | |
| - | <b>Objective:</b> Purify plasmid DNA from cells recently transformed.<br> | + | <html> |
| - | <b>Method:</b> | + | <body> |
| - | <ul> | + | <center> |
| - | <li>Inoculate 5mL of sterile LB liquid media (with 100µg/mL ampicillin) with cells picked from colonies of transformation plates, including the following:<br> | + | <table border="0" width="50%" style="background-color:#000000"> |
| - | Lumazine-dT (J5)<br>
| + | |
| - | pBad-TetR<br>
| + | <tr> |
| - | sRBS (D5,E10)<br></li>
| + | |
| - | <li>NOTE: Lumazine-dT did NOT grow overnight</li> | + | <th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/April"> |
| - | <li>Followed "Boiling Lysis Plasmid Preparation (Miniprep)" protocol. <b>(May 15/2010; JV,TF)</b><br> | + | <img src="https://static.igem.org/mediawiki/2010/8/8a/UofLapril.jpg" width="60"/> |
| - | NOTE: Added 50µL of MilliQ H<sub>2</sub>O (with RNase A at a concentration of 20ng/µL) to dissolve pDNA instead of TE buffer.<br><br></li></ul>
| + | </a> |
| - | <b>Objective:</b> Perform restriction digest on the above prepared plasmid DNA.<br> | + | </th> |
| - | <b>Method:</b><br>
| + | |
| - | Used EcoRI as prefix cutter and PstI as suffix cutter.<br>
| + | <th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/May"> |
| - | Pipetting Scheme for Restriction Tubes:
| + | <img src="https://static.igem.org/mediawiki/2010/7/7b/UofLmaybutton.jpg" width="60"/> |
| - | <table><table border="3"> | + | </a> |
| - | <tr><td><b>Ingredient</b></td><td>Volume/tube (µL)</td><td>Total Volume*</td></tr> | + | </th> |
| - | <tr><td>MilliQ H<sub>2</sub>O</td><td>16</td><td>56</td></tr>
| + | |
| - | <tr><td>Red Buffer (10X)</td><td>2</td><td>7</td></tr> | + | <th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/June"> |
| - | <tr><td>EcoRI</td><td>0.25</td><td>0.875</td></tr> | + | <img src="https://static.igem.org/mediawiki/2010/8/80/UofLjunebutton.jpg" width="60"/> |
| - | <tr><td>PstI</td><td>0.25</td><td>0.875</td></tr> | + | </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> |
| - | *Amount per tube multiplied by 3.5<br>
| + | </center> |
| - | Add 18µL master mix to each plasmid DNA sample<br>
| + | </body> |
| - | Pipetting Scheme for Unrestricted reactions:
| + | </html> |
| - | <table><table border="3">
| + | <hr> |
| - | <tr><td><b>Ingredient</b></td><td>Volume/tube (µL)</td><td>Total Volume*</td></tr>
| + | |
| - | <tr><td>MilliQ H<sub>2</sub>O</td><td>16</td><td>56</td></tr>
| + | <BLOCKQUOTE> |
| - | <tr><td>Red Buffer (10X)</td><td>2</td><td>7</td></tr>
| + | <br> |
| - | </table> | + | |
| - | *Amount per tube multiplied by 3.5<br>
| + | |
| - | Add 18µL master mix to each plasmid DNA sample<br>
| + | |
| - | Buffer Control will be 18µL MilliQ H<sub>2</sub>O + 2µL 10x Red Buffer.<br>
| + | |
| - | Place in 37<sup>o</sup>C water bath at 12:37pm and removed at 1:55pm for approximately 1 hour incubation.<br>
| + | |
| - | Analyze samples on a 1% agarose gel (small gel apparatus).<br>
| + | |
| - | Add 3.3µL of 6x DNA loading dye to each reaction mixture and load.<br>
| + | |
| - | <table><table border="3">
| + | |
| - | <tr><td><b>Lane</td><td>Sample</td><td>Volume Loaded (µL)</td></b></tr>
| + | |
| - | <tr><td>1</td><td>1 kb Ladder</td><td>4</td></tr>
| + | |
| - | <tr><td>2</td><td>Restricted sRBS (E10)</td><td>10</td></tr>
| + | |
| - | <tr><td>3</td><td>sRBS (E10)</td><td>10</td></tr>
| + | |
| - | <tr><td>4</td><td>Restricted sRBS (D5)</td><td>10</td></tr>
| + | |
| - | <tr><td>5</td><td>sRBS (D5)</td><td>10</td></tr>
| + | |
| - | <tr><td>6</td><td>Restricted sRBS-Lumazine-dT</td><td>10</td></tr>
| + | |
| - | <tr><td>7</td><td>sRBS-Lumazine-dT</td><td>10</td></tr>
| + | |
| - | <tr><td>8</td><td>Red Buffer Control</td><td>10</td></tr>
| + | |
| - | </table>
| + | |
| - | Ran gel at 100V for 75 minutes (Start-2:30pm; End-3:45pm)<br>
| + | |
| - | Stained in ethidium bromide for 10 minutes<br><br>
| + | |
| - | <b>Results:</b><br>
| + | |
| - | <b>Picture to come.....</b><br>
| + | |
| - | There is plasmid DNA in each sample which, when cut with both the prefix and suffix enzyme, yields a band approximately 2000bp (size of pSB1A3 is 2157bp).<br>
| + | |
"
