Team:Calgary/Notebook/Protocols/Process4

From 2011.igem.org

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<p>We have a 2X mastermix of taq DNA polymerase, SYBR Green, and dNTPs.  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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<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>
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     <tr>
     <tr>
       <td>Quanta PCR master mix</td>
       <td>Quanta PCR master mix</td>
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       <td>5 uL</td>
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       <td>5 μL</td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td>Primers (Fwd&Rev) at 600nM each</td>
       <td>Primers (Fwd&Rev) at 600nM each</td>
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       <td>3 uL</td>
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       <td>3 μL</td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td>cDNA</td>
       <td>cDNA</td>
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       <td>2 uL</td>
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       <td>2 μL</td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td>Final Volume</td>
       <td>Final Volume</td>
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       <td>10 uL</td>
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       <td>10 μL</td>
     </tr>
     </tr>

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:

qRT-PCR using Quanta PCR mastermix

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.

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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