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Team:Calgary/Notebook/Protocols/Process3
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| - | <li | + | <p>The following protocol is found in the Quanta qScript Reverse Transcription Kit manual, and is used to generate cDNA from isolate RNA (from <i>Pseudomonas spp.</i>) using reverse transcriptase.</p> |
| - | <li | + | <ol> |
| + | <li>Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.</li> | ||
| + | <li>Add the following to a 0.2mL thin-walled PCR tube sitting on ice:</li> | ||
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| + | <CENTER><table border="1px"> | ||
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<tr> | <tr> | ||
<td><b>Component</b></td> | <td><b>Component</b></td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>RNA (40ng – 10ng to 1μg total RNA | + | <td>RNA (40ng – 10ng to 1μg total RNA)</td> |
<td>variable</td> | <td>variable</td> | ||
</tr> | </tr> | ||
| Line 27: | Line 25: | ||
<tr> | <tr> | ||
<td>qScript cDNA synthesis mix</td> | <td>qScript cDNA synthesis mix</td> | ||
| - | <td>4 | + | <td>4 μL</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Final Volume</td> | <td>Final Volume</td> | ||
| - | <td>20 | + | <td>20 μL</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| + | </CENTER> | ||
| - | <li | + | <li>Mix components by gently vortexing and then centrifuge 4s to collect contents.</li> |
| - | <li | + | <li>Incubate in a PCR machine using the following conditions:</li> |
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<tr> | <tr> | ||
</tr> | </tr> | ||
</table> | </table> | ||
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<tr> | <tr> | ||
<td><CENTER>5 min at 25°C</CENTER></td> | <td><CENTER>5 min at 25°C</CENTER></td> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
| - | <li | + | <li>After completion of cDNA synthesis, us 1/20th of the first-strand reaction (2-4μL) for PCR amplification in water. Also do 2,3, and 4-fold dilutions of each sample to ensure that the PCRs will be within the primer linear range. Do 3 replicates of each PCR reaction. Run PCRs on a real-time PCR machine (e.g. ABI 7900).</li> |
| + | <br></br><br></br><br></br> | ||
| + | </ol> | ||
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| + | </html>}} | ||
Latest revision as of 02:59, 29 September 2011
Notebook
Post-Regionals
Journal
- Defining Objectives
- Techniques Workshop
- Bioinformatics & qRT-PCR
- Sensory Element Fishing
- Electrochem - CPR Oxidation
- Electrochem - Reporter Gene
- Pseudomonas Tools
- Microalgae Tools
- NA Viability Assays
- May - July Group Journal
Protocols
Safety
cDNA Synthesis/Reverse Transcription Reaction
The following protocol is found in the Quanta qScript Reverse Transcription Kit manual, and is used to generate cDNA from isolate RNA (from Pseudomonas spp.) using reverse transcriptase.
- Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.
- Add the following to a 0.2mL thin-walled PCR tube sitting on ice:
- Mix components by gently vortexing and then centrifuge 4s to collect contents.
- Incubate in a PCR machine using the following conditions:
- After completion of cDNA synthesis, us 1/20th of the first-strand reaction (2-4μL) for PCR amplification in water. Also do 2,3, and 4-fold dilutions of each sample to ensure that the PCRs will be within the primer linear range. Do 3 replicates of each PCR reaction. Run PCRs on a real-time PCR machine (e.g. ABI 7900).
| Component | Volume |
| RNA (40ng – 10ng to 1μg total RNA) | variable |
| Nuclease free water | variable |
| qScript cDNA synthesis mix | 4 μL |
| Final Volume | 20 μL |
