Team:Lethbridge/Lab Work

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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: @@
-Back to [[Team:Lethbridge/Notebook|Notebook]]<br>
+<div style="background-color:#000000; color:white">
-==April==
+<html>
-===April 13/2010===
-(In the Lab: JV, AS)<br>
-<b>Objective:</b> Test Restriction Endonucleases for Activity<br>
-<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 (&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>
-==May==
+<br>
-===May 5/2010===
-(in the lab: JV)<br>
-<b>Objective:</b> Test Restriction Endonucleases for activity (take 2)<br>
-<b>Relevant Information:</b><br>
-Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks<br>
-Prefix Enzymes are: EcoRI and XbaI<br>
-Suffix Enyzmes are: SpeI and PstI<br>
-(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>
-<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>
-<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>
-<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>
-<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>
-<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>
-</table><br>
-Make up Master Mixes as follows:<br>
-<table><table border="3">
-<tr><td><b>Red MM</td><td>per tube(&micro;L)</td><td>Total*(&micro;L)</td></tr></b>
-<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(&micro;L)</td><td>Total*(&micro;L)</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 3.3&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>
-===May 6/2010===
+<table border="0" width="100%" style="background-color:#000000">
-(in the lab:KG, AS)<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 (&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><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 0.25&micro;L of each enzyme to 19.5&micro;L of master mix<br><br>
-<table><table border ="3">
-<tr><td><b>Tango 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>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>
-*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 0.25&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>
+<tr>
+<th>
+<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>
-===May 10/2010===
-(in the lab:JV)<br>
-<b>Objective:</b> To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis<br><br>
-<b>Method:</b><br>
-<table><table border="3">
-<tr><td><b>Lane</b></td><td>Sample</td><td>Quantity Loaded (&micro;L)</td></tr>
-<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><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>
-<tr><td>5</td><td>SpeI(N)</td><td>10</td></tr>
-<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><td>8</td><td>XbaI+SpeI(O)</td><td>10</td></tr>
-<tr><td>9</td><td>1kb Ladder</td><td>5</td></tr>
 </table>
-Run gel for 60min at 100V<br><br>
-<b>Results:</b><br>
-[[image:100510JRV-EnzymeTest1Cropped.jpg|200px|none]]<br>
-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>Objective:</b>Make 24 LB agar plates with 100&micro;g/mL ampicillin antibiotic.(JV,KG,AV)<br>
-<b>Method:</b>Make 2L of LB media with agar<br>
-x10g Tryptone<br>
-X2.5g Yeast Extract<br>
-x5g NaCl<br>
-x10g 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 500&micro;L of stock ampicillin to 500mL of media<br><br>
-===May 11/2010 Evening===
-(in the lab: KG, AV, MC, TF, JV, JS)</b>
-<b>Objective:</b> To transform the following plasmids into DH5&alpha; <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/Protocols 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>
-===May 12/2010===
+<br>
-(in the lab: JV)<br>
-<b>Objective: Miniprep of plasmid DNA from transformed cells</b>(JV, AV, HB)<br>
+<align="centre">
-<b>Method:</b><br>
+<table border="0"  width="100%"  style="background-color:#000000">
-<ul><li>Inoculate 5mL of LB liquid media (with 100&micro;L/mL Ampicillin) with cells from competent cells plates (picked with sterile toothpick).</li>
-<li>Allow cells in liquid culture to grow overnight in 37<sup>o</sup>C shaking incubator (300RPM)
+<tr>
-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/&micro;L RNase A) instead of TE buffer.</li></ul><br>
+<th>
-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:
-<table><table border="3">
+<a href="https://2010.igem.org/Team:Lethbridge">
-<tr><td>Construct</td><td>Cell in Working Plasmid Box (2010)</td><td>Original Cell in Old Box</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/2/22/UofLHome.jpg" width="80"/>
-<tr><td>sRBS-Lumazine-dT</td><td>A1</td><td>J7</td></tr>
+</a>
-<tr><td>sRNS-Lumazine-dT</td><td>A2</td><td>J8</td></tr>
-<tr><td>CFP Complete</td><td>B6</td><td>D4</td></tr>
+</th>
-<tr><td>Lumazine</td><td>A3</td><td>J4</td></tr>
-<tr><td>pBAD</td><td>A4</td><td>A5</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Team">
-<tr><td>pBAD</td><td>A5</td><td>F10</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/0/0d/UofLTeam.jpg" width="80"/>
-<tr><td>pSB-CEYFP</td><td>B5</td><td></td></tr>
+</a>
-<tr><td>pSB-NEYFP</td><td>B4</td><td></td></tr>
+</th>
-<tr><td>EYFP</td><td>B1</td><td>A4</td></tr>
-<tr><td>N-term tag</td><td>B2</td><td></td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Project">
-</table><br>
+<img src="https://static.igem.org/mediawiki/2010/8/8d/UofLProjectbutton.jpg" width="80"/>
-Also generated sterile glycerol stocks and placed in -80<sup>o</sup>C freezer in the 2010 iGEM box as follows:
+</a>
-<table><table border="3">
+</th>
-<tr><td>Construct</td><td>Cell Working Glycerol Stock Box (2010)</td></tr>
-<tr><td>sRBS-Lumazine-dT (J7)</td><td>B2,C4,D2</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work">
-<tr><td>sRNS-Lumazine-dT (J8)</td><td>C6</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/>
-<tr><td>CFP Complete</td><td>A10, C8</td></tr>
+</a>
-<tr><td>Lumazine</td><td>A8,B10</td></tr>
+</th>
-<tr><td>pBAD (from A5)</td><td>B5,B9</td></tr>
-<tr><td>pBAD (from F10)</td><td>B3,B7</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Parts">
-<tr><td>pSB-CEYFP</td><td>C3,B5</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/8/84/UofLPartsSubmittedToTheRegistrybutton.jpg" width="80"/>
-<tr><td>pSB-NEYFP</td><td>B6,C1</td></tr>
+</a>
-<tr><td>EYFP</td><td>C7,B8</td></tr>
+</th>
-<tr><td>N-term tag</td><td>C2,D4</td></tr>
-</table><br>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Modeling">
-<b>Objective:</b> Restrict plasmid DNA with restriction endonucleases (JV)<br>
+<img src="https://static.igem.org/mediawiki/2010/e/e1/UofLModelingbutton.jpg" width="80"/>
-<b>Method:</b><br>
+</a>
-Have:
+</th>
-lanes of restricted plasmid DNA <br>
-lanes of unrestricted plasmid DNA <br>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Ethics">
-lane of buffer control <br>
+<img src="https://static.igem.org/mediawiki/2010/2/26/UofLEthicsbutton.jpg" width="80"/>
-Use EcoRI (prefix cutter) and PstI (suffix cutter) <br><br>
+</a>
-Pipetting Scheme for Restriction Tubes:
+</th>
-<table><table border="3">
-<tr><td>Ingredient</td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Safety">
-<tr><td>MilliQ H<sub>2</sub>O</td><td>15.5</td><td>155</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/0/00/UofLSafetybutton.jpg" width="80"/>
-<tr><td>Red Buffer (10X)</td><td>2</td><td>20</td></tr>
+</a>
-<tr><td>EcoRI</td><td>0.25</td><td>2.5</td></tr>
+</th>
-<tr><td>PstI</td><td>0.25</td><td>2.5</td></tr>
-</table>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Art">
-*Amount per tube multiplied by 10<br>
+<img src="https://static.igem.org/mediawiki/2010/0/0a/UofLArt.jpg" width="80"/>
-Pipetting Scheme for Unrestricted reactions:
+</a>
-<table><table border="3">
+</th>
-<tr><td><b>Ingredient</b></td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
-<tr><td>MilliQ H<sub>2</sub>O</td><td>16</td><td>160</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/News">
-<tr><td>Red Buffer (10X)</td><td>2</td><td>20</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/c/c3/UofLNewsButton.jpg" width="80"/>
+</a>
+</th>
 </table>
-*Amount per tube multiplied by 10<br>
+</body>
-Buffer Control will be 18&micro;L MilliQ H<sub>2</sub>O + 2&micro;L 10x Red Buffer.<br>
+</html>
-Place in 37<sup>o</sup>C water bath at 2:55pm and removed at 4:57pm for a 2 hour incubation.<br>
+<hr>
-Analyzed restriction digests on a 1% agarose gel (large gel apparatus ~70mL)<br>
-Added 1&micro;L of 6x DNA loading dye to 5&micro;L of sample<br>
-Added 2&micro;L of 6x DNA loading dye to 6&micro;L of TAE buffer and 2&micro;L 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 (&micro;L)</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>
+<html>
-[[image:100513TF.KG.JS-Plasmids_for_Seq.Cropped.jpg|200px|none]]
+<body>
-<b><font size=+2>May 13/2010 Evening</font>(in lab: AS,TF,KG,JS,MC)</b><br>
+<center>
-<b>Objective:</b> To make a second attempt at transforming plasmids that didn't transform the first time. These plasmids are:<br>
+<table border="0"  width="28%"  style="background-color:#000000">
-<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 DH5&alpha; cells were used up in the last transformation, had to aliquot an additional 50x 20&micro;L aliquots (MC,TF)<br>
-Transform plasmid DNA (Using "Competent Cell Transformation" Protocol) into newly aliquotted DH5&alpha; 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 250&micro;L 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>
-===May 14/2010===
+<tr>
-(in the lab: JV)<br>
+<th>
-<b>Objective:</b> Quantify pDNA concentration in order to ensure sufficient material for sequence analysis.<br>
+<div class="miniBar">
-<b>Method:</b> Measure absorbance of samples at 260nm.<br>
+		<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>
-<b>Results:</b><br>
+		<div class="miniContainer">
-<table><table border="3">
-<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>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work">
-<b>Method:</b>
+<img src="https://static.igem.org/mediawiki/2010/7/73/UofLNotebookbutton.jpg" width="80"/>
-<ul>
+</a>
-<li>Inoculate 5mL of sterile LB liquid media (with 100&micro;g/mL ampicillin) with cells picked from colonies of transformation plates, including the following:<br>
+</th>
-Lumazine-dT (J5)<br>
-pBad-TetR<br>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols">
-sRBS (D5,E10)<br></li>
+<img src="https://static.igem.org/mediawiki/2010/9/91/UofLprotocolsbutton.jpg" width="60"/>
-<li>NOTE: Lumazine-dT did NOT grow overnight</li>
+</a>
-<li>Followed "Boiling Lysis Plasmid Preparation (Miniprep)" protocol. <b>(May 15/2010; JV,TF)</b><br>
+</th>
-NOTE: Added 50&micro;L of MilliQ H<sub>2</sub>O (with RNase A at a concentration of 20ng/&micro;L) to dissolve pDNA instead of TE buffer.<br><br></li></ul>
-<b>Objective:</b> Perform restriction digest on the above prepared plasmid DNA.<br>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Calendar">
-<b>Method:</b><br>
+<img src="https://static.igem.org/mediawiki/2010/7/73/UofLcalendar.jpg" width="60"/>
-Used EcoRI as prefix cutter and PstI as suffix cutter.<br>
+</a>
-Pipetting Scheme for Restriction Tubes:
+</th>
-<table><table border="3">
-<tr><td><b>Ingredient</b></td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
+<th>
-<tr><td>MilliQ H<sub>2</sub>O</td><td>16</td><td>56</td></tr>
+<div class="miniBar">
-<tr><td>Red Buffer (10X)</td><td>2</td><td>7</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>EcoRI</td><td>0.25</td><td>0.875</td></tr>
+		<div class="miniContainer">
-<tr><td>PstI</td><td>0.25</td><td>0.875</td></tr>
+</th>
-</table>
+<tr>
-*Amount per tube multiplied by 3.5<br>
-Add 18&micro;L master mix to each plasmid DNA sample<br>
-Pipetting Scheme for Unrestricted reactions:
-<table><table border="3">
-<tr><td><b>Ingredient</b></td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
-<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>
-</table>
-*Amount per tube multiplied by 3.5<br>
-Add 18&micro;L master mix to each plasmid DNA sample<br>
-Buffer Control will be 18&micro;L MilliQ H<sub>2</sub>O + 2&micro;L 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 3.3&micro;L 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 (&micro;L)</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>
+</center>
-Stained in ethidium bromide for 10 minutes<br><br>
+</body>
-<b>Results:</b><br>
+</html>
-[[image:100515JV.jpg|200px|none]]<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><br>
-===May 17/2010===
+<hr>
-(in the lab: JV, AV)<br>
-Make agar plates with 100&micro;g/mL of ampicillin<br>
-Make 5 x 5mL sterile liquid SOC broth<br>
-Make 13 x 5mL sterile liquid LB broth<br><br>
-===May 17/2010 Evening===
+<html>
-(in the lab: TF, AS)<br>
+<center>
-<b>Objective:</b> To grow cells for future use<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!
-Streaked plates from glycerol stocks of the following:<br>
+</center>
-<u>iGEM 2007 -80<sup>o</sup>C Freezer Box:</u>
+</html>
-<table><table border="3">
-<tr><td>Construct</td><td>Cell type</td><td>Location of cells</td></tr>
+<html>
-<tr><td>xylE</td><td>DH5&alpha;</td><td>C4</td></tr>
+<body>
-<tr><td>xylE</td><td>BL21(DE3)</td><td>B4</td></tr>
+<center>
-<tr><td>C-term Bba</td><td>DH5&alpha;</td><td>H4</td></tr>
+<table border="0"  width="50%"  style="background-color:#000000">
-<tr><td>C-term Bba</td><td>DH5&alpha;</td><td>I4</td></tr>
-<tr><td>C-term Bba</td><td>DH5&alpha;</td><td>J4</td></tr>
+<tr>
-<tr><td>Mr. Gene mms6</td><td>DH5&alpha;</td><td>A6</td></tr>
-<tr><td>Mr. Gene mms6</td><td>DH5&alpha;</td><td>B6</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/April">
-</table><br>
+<img src="https://static.igem.org/mediawiki/2010/8/8a/UofLapril.jpg" width="60"/>
-<u>iGEM 2010 -80<sup>o</sup>C Freezer Box:</u>
+</a>
-<table><table border="3">
+</th>
-<tr><td>Construct</td><td>Cell type</td><td>Location of cells</td></tr>
-<tr><td>pLacI</td><td>DH5&alpha;</td><td>B1</td></tr>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/May">
-<tr><td>dT</td><td>BL21(DE3)</td><td>D1</td></tr>
+<img src="https://static.igem.org/mediawiki/2010/7/7b/UofLmaybutton.jpg" width="60"/>
-</table><br>
+</a>
-Incubated at 37<sup>o</sup>C, beginning at 20h00 (8:00pm)<br><br>
+</th>
-Made liquid cultures from cells taken from transformation plates (grown on May 13/2010):
-<ul>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/June">
-<li>sRBS (D5)</li>
+<img src="https://static.igem.org/mediawiki/2010/8/80/UofLjunebutton.jpg" width="60"/>
-<li>sRNB (E10)</li>
+</a>
-<li>Lumazine-dT (1)</li>
+</th>
-<li>pBad-TetR</li></ul>
-Incubated at 37<sup>o</sup>C, beginning at 20h30 (8:30pm); shaking at 300RPM<br>
+<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/July">
-<b>Results:</b><br>
+<img src="https://static.igem.org/mediawiki/2010/5/53/UofLjulybutton.jpg" width="60"/>
-All streak plates (with cells from glycerol stocks) grew.<br>
+</a>
-Only sRBS (D5) and pBad-TetR cells (from transformation plates) grew.<br>
+</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>
+</center>
+</body>
+</html>
+<hr>
-===May 18/2010===
+<BLOCKQUOTE>
-(in the lab: JV, AV, HB)<br>
+<br>
-<b>NOTE:</b> Cells from liquid cultures grown last night (May 17/2010) were made into glycerol stocks and placed into the [[Team:Lethbridge/Notebook/Working_Glycerol_Stocks|working glycerol stock box]] as follows:<br>
-<ul>
-<li>pBAD-TetR - E5</li>
-<li>pBAD-TetR - E6</li>
-<li>sRBS (D5) - E7</li>
-<li>sRBS (D5) - E8</li></ul><br>
-<b>Objective:</b> To isolate plasmid DNA of pBad-TetR and sRBS (D5) and cut with restriction enzymes.<br><br>
-<b>Method:</b><br>
-Use [[Team:Lethbridge/Notebook/Protocols|boiling lysis miniprep]] to prepare plasmid DNA.
-Digest sRBS with PstI only; digest pBAD-TetR with SpeI (old and new) and PstI.<br><br>
-Reaction conditions for PstI using Orange Buffer.<br>
-<table><table border="3">
-<tr><td>Ingredient</td><td>Volume/tube (&micro;L)</td></tr>
-<tr><td>Milli-Q H<sub>2</sub>O</td><td>15.75</td></tr>
-<tr><td>Orange Buffer</td><td>2</td></tr>
-<tr><td>Plasmid DNA</td><td>2</td></tr>
-<tr><td>PstI</td><td>0.25</td></tr></table><br>
-Reaction conditions for PstI using Tango Buffer.<br>
-<table><table border="3">
-<tr><td>Ingredient</td><td>Volume/tube (&micro;L)</td></tr>
-<tr><td>Milli-Q H<sub>2</sub>O</td><td>15.75</td></tr>
-<tr><td>Tango Buffer</td><td>2</td></tr>
-<tr><td>Plasmid DNA</td><td>2</td></tr>
-<tr><td>PstI</td><td>0.25</td></tr></table><br>