Team:Lethbridge/Notebook/Lab Work/August
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==<font color="white">Aug 9/2010== | ==<font color="white">Aug 9/2010== | ||
- | + | (In Lab: JV)<br> | |
- | <b> Objective:</b> Prepared glycerol stocks & Miniprepped the following using the Qiagen Protocol | + | <b> Objective:</b> Prepared glycerol stocks & Miniprepped the following using the Qiagen Protocol.<br> |
<table border ="3"> | <table border ="3"> | ||
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<tr><td>mRBS-TetR Colony 3 | <tr><td>mRBS-TetR Colony 3 | ||
</table><br> | </table><br> | ||
+ | |||
+ | |||
+ | <b>Objective:</b> To determine which of the previous ligations worked.<br> | ||
+ | |||
+ | <b>Method:</b> Restrict with single cutter and double cutter then run on 2% agarose gel. <br> | ||
+ | |||
+ | |||
+ | Restriction Reaction (SINGLE) | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Ingredient</b></td><td>Volume(µL)</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75</td></tr> | ||
+ | <tr><td>Orange Buffer (10x)</td><td>2</td></tr> | ||
+ | <tr><td>pDNA</td><td>2</td></tr> | ||
+ | <tr><td>EcoRI</td><td>0.25</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | Restriction Reaction (DOUBLE) | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Ingredient</b></td><td>Volume(µL)</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.50</td></tr> | ||
+ | <tr><td>Orange Buffer (10x)</td><td>2</td></tr> | ||
+ | <tr><td>pDNA</td><td>2</td></tr> | ||
+ | <tr><td>EcoRI</td><td>0.25</td></tr> | ||
+ | <tr><td>PstI</td><td>0.25</td></tr> | ||
+ | </table> | ||
+ | |||
+ | Unrestricted Control | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Ingredient</b></td><td>Volume(µL)</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>0 Water</td><td>16</td></tr> | ||
+ | <tr><td>Orange Buffer (10x)</td><td>2</td></tr> | ||
+ | <tr><td>pDNA </td><td>2</td></tr> | ||
+ | </table> <br> | ||
+ | |||
+ | DNA was restricted for 1 hour at 37<sup>o</sup>C.<br> | ||
+ | |||
+ | Analyzed results on a 2% agarose gel. Load order as follows:<br> | ||
+ | |||
+ | Ran gel at 100V for 40 minutes.<br> | ||
+ | |||
+ | <b>Results:</b> <br> | ||
+ | |||
+ | |||
+ | <b>Conclusions:</b> Plasmid DNA prep and restriction was successful.<br><br> | ||
+ | |||
+ | <b>Objective:</b> Ligate rbs-xylE (Bba_J33204) to our double terminator, and insert it into the pSB1T3 plasmid backbone.<br> | ||
+ | <b>Method:</b><br> | ||
+ | <ul> | ||
+ | <li><u>Restrictions</u><br> | ||
+ | *Restrict rbs-xylE wit EcoRI and SpeI (Red Buffer)<br> | ||
+ | *Restrict the double terminator with XbaI and PstI (Tango Buffer)<br> | ||
+ | *Restrict pSB1T3 with EcoRI and PstI (Red Buffer)<br> | ||
+ | Set up reactions as follows:<br> | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Component</b></td><td><b>Volume (µL)</b></td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>O</td><td>15.5</td></tr> | ||
+ | <tr><td>Buffer</td><td>2</td></tr> | ||
+ | <tr><td>pDNA</td><td>2</td></tr> | ||
+ | <tr><td>Enzyme</td><td>0.25 + 0.25</td></tr></table> | ||
+ | |||
+ | Set up control reaction as follows: | ||
+ | *MilliQ H<sub>2</sub>O - 16µL<br> | ||
+ | *Buffer - 2µL<br> | ||
+ | *pDNA - 2µL<br> | ||
+ | Incubated reactions for 65 minutes at 37<sup>o</sup>C<br> | ||
+ | Killed enzymes by incubating reactions for 10 minutes at 65<sup>o</sup>C<br> | ||
+ | |||
+ | <li><u>Ligation</u><br> | ||
+ | Reaction set up as follows: | ||
+ | *T4 DNA ligase - 0.25µL<br> | ||
+ | *rbs-xylE - 5µL<br> | ||
+ | *dT - 3µL<br> | ||
+ | *pSB1T3 - 8µL<br> | ||
+ | *10x Ligation Buffer - 2µL<br> | ||
+ | *MilliQ H<sub>2</sub>O - 1.75µL<br> | ||
+ | Incubated reactions overnight at room temperature (total of 19.5 hours)<br> | ||
+ | Killed enzymes by incubating reactions for 10 minutes at 80<sup>o</sup>C</ul> |