Team:Lethbridge
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<b><font size=+2>May 6/2010</font>(in the lab:KG, AS)</b><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. | + | <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> | ||
+ | *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> | ||
+ | <b>Method:</b><br> | ||
+ | <table><table border="3"> | ||
+ | <tr><td><b>Lane</b></td><td>Sample</td><td>Quantity Loaded (µL)</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> | ||
Revision as of 00:20, 15 May 2010
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April
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April 13/2010 (In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available
April
May
April 13/2010 (In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available