Team:Calgary/Notebook/Protocols/Process4
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- | <p>We have a mastermix of | + | <p>We have a 2X mastermix of taq DNA polymerase, SYBR Green, and dNTPs. Primers are stored at 100μM and used at final concentrations between 200-600nM. The PCR mix will be as follows:</p> |
+ | <CENTER> | ||
<tr> | <tr> | ||
<td><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="qRT-PCR"></a> qRT-PCR using Quanta PCR mastermix </p></div></td> | <td><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="qRT-PCR"></a> qRT-PCR using Quanta PCR mastermix </p></div></td> | ||
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<tr> | <tr> | ||
<td>Quanta PCR master mix</td> | <td>Quanta PCR master mix</td> | ||
- | <td>5 | + | <td>5 μL</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Primers (Fwd&Rev) at 600nM each</td> | <td>Primers (Fwd&Rev) at 600nM each</td> | ||
- | <td>3 | + | <td>3 μL</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>cDNA</td> | <td>cDNA</td> | ||
- | <td>2 | + | <td>2 μL</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Final Volume</td> | <td>Final Volume</td> | ||
- | <td>10 | + | <td>10 μL</td> |
</tr> | </tr> | ||
</table> | </table> | ||
+ | </CENTER> | ||
<p>The following cylcing conditions are used.</p> | <p>The following cylcing conditions are used.</p> | ||
- | + | <CENTER> <table border="1px"> | |
<tr> | <tr> | ||
- | + | <p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="reaction conditions"></a> </p></div> | |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
<td><CENTER>2 min at 50°C</CENTER></td> | <td><CENTER>2 min at 50°C</CENTER></td> | ||
</tr> | </tr> | ||
+ | |||
<tr> | <tr> | ||
<td><CENTER>10 min at 95°C</CENTER></td> | <td><CENTER>10 min at 95°C</CENTER></td> | ||
</tr> | </tr> | ||
+ | |||
<tr><td><b>40 cycles of:</b></tr></td> | <tr><td><b>40 cycles of:</b></tr></td> | ||
- | <tr> | + | |
+ | <tr> | ||
<td><CENTER>15 sec at 95°C</CENTER></td> | <td><CENTER>15 sec at 95°C</CENTER></td> | ||
</tr> | </tr> | ||
- | + | ||
+ | <tr> | ||
<td><CENTER>1 min at 60°C</CENTER></td> | <td><CENTER>1 min at 60°C</CENTER></td> | ||
</tr> | </tr> | ||
- | + | ||
+ | <tr> | ||
<td><b>Melting curve analysis</b></td> | <td><b>Melting curve analysis</b></td> | ||
</tr> | </tr> | ||
- | <tr> | + | |
+ | <tr> | ||
<td><CENTER>15 sec at 95°C</CENTER></td> | <td><CENTER>15 sec at 95°C</CENTER></td> | ||
</tr> | </tr> | ||
- | </table></CENTER></html>}} | + | |
+ | </table> | ||
+ | </CENTER> | ||
+ | </html>}} |
Latest revision as of 22:04, 27 September 2011
qRT-PCR
We have a 2X mastermix of taq DNA polymerase, SYBR Green, and dNTPs. Primers are stored at 100μM and used at final concentrations between 200-600nM. The PCR mix will be as follows:
Component | Volume |
Quanta PCR master mix | 5 μL |
Primers (Fwd&Rev) at 600nM each | 3 μL |
cDNA | 2 μL |
Final Volume | 10 μL |
The following cylcing conditions are used.
40 cycles of: |
Melting curve analysis |