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Team:Lethbridge/Notebook/Lab Work/April
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| - | April | + | Back to [[Team:Lethbridge/Notebook|Notebook]]<br> |
| + | Back to [[Team:Lethbridge/Notebook/Lab_Work|Lab Work]] | ||
| + | ==April== | ||
| + | ===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 (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> | ||
Revision as of 02:52, 2 June 2010
Back to Notebook
Back to Lab Work
April
April 13/2010
(In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available
| Endonuclease | Optimal Buffer(**) | Other Buffers |
| EcoRV | None | 2xT(100%); O,G(50-100%) |
| EcoRI | Red | O(100%);R(100%)(*);2xT(100%) |
| BcuI/SpeI | Tango | B(50-100%);G(50-100%) |
| XbaI | Tango | B,G,2xT(50-100%) |
| PstI | Orange | R(100%); B,G,T,2xT(50-100%) |
| DpnI | Tango | B,G(100%): O,R,2xT(50-100%) |
(*)Star Activity
(**)Optimal Buffer from Fermentas
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
Red Buffer: EcoRI, PstI, Control (No Enzyme)
Tango Buffer: BcuI/SpeI, XbaI, DpnI, Control (No Enzyme>
Methods:
Set up Master Mixes:
| Red MM | per tube (µL) | Total (µL) |
| MilliQ H20 | 13.75 | 55 |
| Red Buffer (10x) | 2 | 7 |
| pUC19 (10pg/µL) | 2 | 7 |
| Total | 19.75 | 69 |
| Tango MM | per tube (µL) | Total (µL) |
| MilliQ H20 | 13.75 | 55 |
| Tango Buffer (10x) | 2 | 7 |
| pUC19 (10pg/µL) | 2 | 7 |
| Total | 19.75 | 69 |
To each tube, add 19.75µL of master mix and 0.25µL of enzyme
Incubated reaction mixes at 37oC (Start:7:00pm; End:7:45pm)
Add 3.3µL of 6x loading dye to each reaction mixture and load 10µL final volume onto a 1% agarose (in TAE) gel.
Add 1µL of 6x loading dye to 1µL of GeneRuler 1kb ladder (at 0.5µg/µL)
Gel loading order as follows:
| Lane | Sample |
| 1 | 1kb Ladder |
| 2 | Tango Control |
| 3 | DpnI (Tango) |
| 4 | BcuI/SpeI (Tango) |
| 5 | XbaI (Tango) |
| 6 | EcoRI (Red) |
| 7 | PstI (Red) |
| 8 | Red Control |
| 9 | Empty |
| 10 | Empty |
Ran gel at 100V for 1 hour
Results: pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain).
