Team:USTC CHINA/Notebook/Protocols/PCR

From 2013.igem.org

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<h1>PCR</h1>
<h1>PCR</h1>
<p>Performed with TaKaRa PrimeSTAR® GXL DNA Polymerase</br>
<p>Performed with TaKaRa PrimeSTAR® GXL DNA Polymerase</br>
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</br>
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<table width="580" border="2">
<table width="580" border="2">
   <tr>
   <tr>
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3. Gently vortex the samples and spin down.</br>
3. Gently vortex the samples and spin down.</br>
4. Perform PCR using recommended thermal cycling conditions:</br>
4. Perform PCR using recommended thermal cycling conditions:</br>
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<table width="580" border="2">
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  <tr>
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    <td>Step</td>
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    <td>Temperature, °C</td>
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    <td>Time</td>
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    <td>Number of cycles</td>
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  </tr>
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  <tr>
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    <td>Initial denaturation</td>
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    <td>98</td>
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    <td>8 min</td>
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    <td>1</td>
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  </tr>
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  <tr>
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    <td>Denaturation</td>
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    <td>98</td>
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    <td>10 s</td>
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    <td>25~40</td>
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  </tr>
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  <tr>
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    <td>Annealing</td>
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    <td>55(if Tm<60)</td>
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    <td>15 s</td>
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    <td>25~40</td>
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  </tr>
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  <tr>
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    <td>Annealing</td>
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    <td>60(if Tm>60)</td>
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    <td>15 s</td>
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    <td>25~40</td>
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  </tr>
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  <tr>
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    <td>Extension</td>
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    <td>68</td>
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    <td>1 min/kb</td>
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    <td>25~40</td>
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  </tr>
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  <tr>
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    <td>Final Extension</td>
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    <td>68</td>
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    <td>5-15 min</td>
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    <td>1</td>
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  </tr>
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</table>
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</br>
</p></div>
</p></div>

Revision as of 17:04, 25 September 2013

PCR

Performed with TaKaRa PrimeSTAR® GXL DNA Polymerase

Volume (μl)
5×PrimeSTAR GXL Buffer 10
dNTP Mixture（2.5 mM each） 4
Forward primer 10~15 pmol
Reverse primer 10~15 pmol
Template DNA 10pg~10ng
PrimeSTAR GXL DNA Polymerase 1
ddH2O To 50
Total 50

3. Gently vortex the samples and spin down.
4. Perform PCR using recommended thermal cycling conditions:
Step Temperature, °C Time Number of cycles
Initial denaturation 98 8 min 1
Denaturation 98 10 s 25~40
Annealing 55(if Tm<60) 15 s 25~40
Annealing 60(if Tm>60) 15 s 25~40
Extension 68 1 min/kb 25~40
Final Extension 68 5-15 min 1

notebook

Retrieved from "http://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/PCR"