Team:Lethbridge/Notebook/Lab Work/April
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+ | <th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/April"> | ||
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+ | <BLOCKQUOTE> | ||
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+ | =<font color="white">April= | ||
+ | ==<font color="white">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><b>Endonuclease</b></td><td><b>Optimal Buffer(**)</b></td><td><b>Other Buffers</b></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> | ||
+ | <b>Methods:</b> | ||
+ | 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> |