Difference between revisions of "Team:Arizona State/Experiments"
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<p>PCR is used to rapidly amplify certain segments of DNA that are selected via designed primers. This protocol was used to accomplish a variety of goals, from extraction of parts from a cloning vector to the addition of restriction cut sites at the ends of parts. </p> | <p>PCR is used to rapidly amplify certain segments of DNA that are selected via designed primers. This protocol was used to accomplish a variety of goals, from extraction of parts from a cloning vector to the addition of restriction cut sites at the ends of parts. </p> | ||
<ol> | <ol> | ||
| − | <li>Defrost template, | + | <li>Defrost template, 2x Master Mix and primers |
| − | <li>Determine desired total working volume (~ | + | <li>Determine desired total working volume (~25uL) |
| + | <li>Label reaction tubes | ||
</ol> | </ol> | ||
| + | <table style="width:100%"> | ||
| + | <tr> | ||
| + | <th>Part</th> | ||
| + | <th>Concentration</th> | ||
| + | <th>Volume for 25uL reaction</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>2x Master Mix</td> | ||
| + | <td>2x</td> | ||
| + | <td>12.5uL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>dsDNA Template</td> | ||
| + | <td>-</td> | ||
| + | <td>1uL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Forward primer</td> | ||
| + | <td>-</td> | ||
| + | <td>1uL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Reverse primer</td> | ||
| + | <td>-</td> | ||
| + | <td>1uL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>DI H2O</td> | ||
| + | <td>-</td> | ||
| + | <td>9.5uL</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <h4>Sample Reaction</h4> | ||
| + | <p>This is an example protocol used in the thermal cycler, durations and temperatures subject to change</p> | ||
| + | <br> | ||
| + | <table style="width:100%"> | ||
| + | <tr> | ||
| + | <th>Step Name</th> | ||
| + | <th># of Cycles</th> | ||
| + | <th>Temperature(℃)</th> | ||
| + | <th>Duration(min)</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Initial</td> | ||
| + | <td>1</td> | ||
| + | <td>98</td> | ||
| + | <td>3</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Denaturation</td> | ||
| + | <td>30</td> | ||
| + | <td>98</td> | ||
| + | <td>0.5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Annealing</td> | ||
| + | <td>30</td> | ||
| + | <td>56</td> | ||
| + | <td>0.5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Elongation</td> | ||
| + | <td>30</td> | ||
| + | <td>72</td> | ||
| + | <td>0.5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Final Elongation</td> | ||
| + | <td>1</td> | ||
| + | <td>72</td> | ||
| + | <td>10</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Storage</td> | ||
| + | <td>1</td> | ||
| + | <td>4</td> | ||
| + | <td>∞</td> | ||
| + | </tr> | ||
| + | </table> | ||
</div> | </div> | ||
<p> | <p> | ||
Revision as of 10:06, 31 August 2016
Protocols
[1A] Polymerase Chain Reaction
PCR is used to rapidly amplify certain segments of DNA that are selected via designed primers. This protocol was used to accomplish a variety of goals, from extraction of parts from a cloning vector to the addition of restriction cut sites at the ends of parts.
- Defrost template, 2x Master Mix and primers
- Determine desired total working volume (~25uL)
- Label reaction tubes
| Part | Concentration | Volume for 25uL reaction |
|---|---|---|
| 2x Master Mix | 2x | 12.5uL |
| dsDNA Template | - | 1uL |
| Forward primer | - | 1uL |
| Reverse primer | - | 1uL |
| DI H2O | - | 9.5uL |
Sample Reaction
This is an example protocol used in the thermal cycler, durations and temperatures subject to change
| Step Name | # of Cycles | Temperature(℃) | Duration(min) |
|---|---|---|---|
| Initial | 1 | 98 | 3 |
| Denaturation | 30 | 98 | 0.5 |
| Annealing | 30 | 56 | 0.5 |
| Elongation | 30 | 72 | 0.5 |
| Final Elongation | 1 | 72 | 10 |
| Storage | 1 | 4 | ∞ |
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project
