Team:Calgary/Notebook/Protocols/Process4

From 2011.igem.org

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<p>We have a mastermix of PCR enzyme, sybr green, and dNTPs that is 2x.  Primers will be stored at 100μM and used at final concentrations between 200-600nM.  The PCR mix will be as follows:</p>
<p>We have a mastermix of PCR enzyme, sybr green, and dNTPs that is 2x.  Primers will be stored at 100μM and used at final concentrations between 200-600nM.  The PCR mix will be as follows:</p>
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       <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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<p>The following cylcing conditions are used.</p>
<p>The following cylcing conditions are used.</p>
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      <td><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="reaction conditions"></a> </p></div></td>
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    <p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="reaction conditions"></a> </p></div>
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Revision as of 21:57, 27 September 2011


qRT-PCR

We have a mastermix of PCR enzyme, sybr green, and dNTPs that is 2x. Primers will be stored at 100μM and used at final concentrations between 200-600nM. The PCR mix will be as follows:

qRT-PCR using Quanta PCR mastermix

Component Volume
Quanta PCR master mix 5 uL
Primers (Fwd&Rev) at 600nM each 3 uL
cDNA 2 uL
Final Volume 10 uL

The following cylcing conditions are used.

2 min at 50°C
10 min at 95°C
40 cycles of:
15 sec at 95°C
1 min at 60°C
Melting curve analysis
15 sec at 95°C

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