Team:USTC CHINA/Notebook/Protocols/PCR

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<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a></div></div>
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<h1>Gel Extraction</h1>
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<h1>PCR</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p>Performed with TaKaRa PrimeSTAR® GXL DNA Polymerase</br>
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Protocol
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1. Excise gel slice containing DNA fragment of interest.
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<table width="580" border="2">
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2. Add 3×sample volume of Buffer DE-A.
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  <tr>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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    <td></td>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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    <td>Volume (μl)</td>
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<img src="https://static.igem.org/mediawiki/2013/4/4d/2013igemustc-china_Protocol1.png" width="580" height="200" />  
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  </tr>
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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  <tr>
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    <td>5×PrimeSTAR GXL Buffer</td>
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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    <td>10</td>
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Repeat wash with Buffer W2
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  </tr>
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Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
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  <tr>
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    <td>dNTP Mixture（2.5 mM each）</td>
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5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
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    <td>4</td>
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  </tr>
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  <tr>
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    <td>Forward primer</td>
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    <td>10~15 pmol</td>
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  </tr>
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  <tr>
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    <td>Reverse primer</td>
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    <td>10~15 pmol</td>
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  </tr>
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  <tr>
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    <td>Template DNA</td>
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    <td>10pg~10ng</td>
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  </tr>
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  <tr>
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    <td>PrimeSTAR GXL DNA Polymerase</td>
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    <td>1</td>
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  </tr>
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  <tr>
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    <td>ddH<sub>2</sub>O</td>
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    <td>To 50</td>
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  </tr>
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  <tr>
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    <td>Total</td>
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    <td>50</td>
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  </tr>
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 +
</table>
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</br>
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3. Gently vortex the samples and spin down.</br>
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4. Perform PCR using recommended thermal cycling conditions:</br>
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</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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 +
 
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</table>
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</br>
</p></div>
</p></div>
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Latest revision as of 08:57, 27 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"