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, 2x Master Mix and primers | + | <li>Defrost template, 2x Master Mix and primers</li> |
| − | <li>Determine desired total working volume (~25uL) | + | <li>Determine desired total working volume (~25uL)</li> |
| − | <li>Label reaction tubes | + | <li>Label reaction tubes</li> |
</ol> | </ol> | ||
| Line 50: | Line 50: | ||
<h4>Sample Reaction</h4> | <h4>Sample Reaction</h4> | ||
<p>This is an example protocol used in the thermal cycler, durations and temperatures subject to change</p> | <p>This is an example protocol used in the thermal cycler, durations and temperatures subject to change</p> | ||
| − | |||
<table style="width:100%"> | <table style="width:100%"> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
| + | </div> | ||
| + | <div class="container"> | ||
| + | <h3>[1B] PCR Cleanup </h3> | ||
| + | <p>The Qiagen Qiaquick PCR cleanup kit was used to purify the thermal cycler product and remove remaining polymerases and nucleotides.</p> | ||
| + | <ol> | ||
| + | <li>Gather the solutions from the PCR cleanup kit</li> | ||
| + | <li>Mix 5 times volume of Buffer PB with PCR reaction tube</li> | ||
| + | <li>Add 10uL 3M sodium acetate</li> | ||
| + | <li>Spin down the mixture in a QIAquick filter column at max speed for 60s. Discard flow-through and replace column into tube </li> | ||
| + | <li>Add 750uL of Buffer PE to the mixture and spin down at max speed for 60s to wash. Discard flow-through and replace column</li> | ||
| + | <li>Centrifuge the tube one more time to remove any remaining wash buffer</li> | ||
| + | <li>Place column in clean 1.5mL plastic tube</li> | ||
| + | <li>Add 30uL of Buffer EB to the column and let stand for 1min. Centrifuge at max speed for 1 min. </li> | ||
| + | </ol> | ||
</div> | </div> | ||
<p> | <p> | ||
Revision as of 09:31, 1 September 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 | ∞ |
[1B] PCR Cleanup
The Qiagen Qiaquick PCR cleanup kit was used to purify the thermal cycler product and remove remaining polymerases and nucleotides.
- Gather the solutions from the PCR cleanup kit
- Mix 5 times volume of Buffer PB with PCR reaction tube
- Add 10uL 3M sodium acetate
- Spin down the mixture in a QIAquick filter column at max speed for 60s. Discard flow-through and replace column into tube
- Add 750uL of Buffer PE to the mixture and spin down at max speed for 60s to wash. Discard flow-through and replace column
- Centrifuge the tube one more time to remove any remaining wash buffer
- Place column in clean 1.5mL plastic tube
- Add 30uL of Buffer EB to the column and let stand for 1min. Centrifuge at max speed for 1 min.
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project
