Difference between revisions of "Team:Austin UTexas LASA/Experiments"
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| + | <p>Reagents and protocol from Zymo DNA Clean & Concentrator Kit</p> | ||
| + | <br> | ||
| + | <p><strong>Reagents</strong></p> | ||
<table> | <table> | ||
<tr> | <tr> | ||
| − | <th> | + | <th>DNA Binding Buffer</th> |
| − | <th>5 uL</th> | + | <th>Refer to table below.</th> |
| + | </tr> | ||
| + | <tr> | ||
| + | <th>DNA Wash Buffer</th> | ||
| + | <th>Refer to table below.</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <th>NF H2O</th> | ||
| + | <th>Volume dependent on objective.</th> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <br> | ||
| + | <p><strong>Protocol:</strong>all centrifugation steps should be performed between 10,000 - 16,000g. | ||
| + | <br> | ||
| + | <ol> | ||
| + | <li>On ice, add DNA Binding Buffer (2-7 volumes) and the sample (see table below) in a microcentrifuge tube and mix briefly by vortexing.</li> | ||
| + | <table> | ||
| + | <th><strong>Application</strong></th> | ||
| + | <th><strong>DNA Binding Buffer: Sample</strong></th> | ||
| + | <th><strong>Example</strong></th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <th>Genomic DNA (> 2 kb)</th> | ||
| + | <th>2 : 1</th> | ||
| + | <th>200 uL : 100 uL</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <th>PCR Product, DNA Fragment</th> | ||
| + | <th>5 : 1</th> | ||
| + | <th>500 uL : 100 uL</th> | ||
</tr> | </tr> | ||
</table> | </table> | ||
| − | + | <li>Transfer mixture to a minicolumn inside of a collection tube and centrifuge for 30 seconds, discard flow-through.</li> | |
| + | <li>Add 200 uL DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat 1x. </li> | ||
| + | <li>Add water (volume dependent on objective) directly to the column, and incubate at room temperature for 1 min.</li> | ||
| + | <li>Transfer the column to a clean microcentrifuge tube and centrifuge for 1 min to elute the DNA.</li> | ||
| + | </ol> | ||
| + | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 22:31, 31 October 2017
Experiments
Reactants
| 10x Reaction Mix | 5 uL |
|---|---|
| For primer (10 uM) | 1 uL |
| Rev primer (10 uM) | 1 uL |
| Template | 1 uL |
| Taq Polymerase | 1 uL |
| MgCl2 (25 mM) | 1 uL |
| DMSO | 1 uL |
| NF H2o | 41 uL |
| Total | 50 uL |
Protocol
- On ice, add all reagents to PCR tube, Taq polymerase last.
- Run thermocycler program:
- 95 C for 5 min (2 min if NOT using Hotstart)
> 10 cycles:
95 C for 20 s
0.3 C/s to 50 C
72 C for # s (extension = 1 min/kb)
> 15 cycles:
95 C for 20 s
55 C for 20 s
72 C for 1 min
72 C for 10 min
4 C forever
Reagents and protocol from Zymo DNA Clean & Concentrator Kit
Reagents
| DNA Binding Buffer | Refer to table below. |
|---|---|
| DNA Wash Buffer | Refer to table below. |
| NF H2O | Volume dependent on objective. |
Protocol:all centrifugation steps should be performed between 10,000 - 16,000g.
- On ice, add DNA Binding Buffer (2-7 volumes) and the sample (see table below) in a microcentrifuge tube and mix briefly by vortexing.
- Transfer mixture to a minicolumn inside of a collection tube and centrifuge for 30 seconds, discard flow-through.
- Add 200 uL DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat 1x.
- Add water (volume dependent on objective) directly to the column, and incubate at room temperature for 1 min.
- Transfer the column to a clean microcentrifuge tube and centrifuge for 1 min to elute the DNA.
| Application | DNA Binding Buffer: Sample | Example |
|---|---|---|
| Genomic DNA (> 2 kb) | 2 : 1 | 200 uL : 100 uL |
| PCR Product, DNA Fragment | 5 : 1 | 500 uL : 100 uL |
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