Team:USTC CHINA/Notebook/Protocols/Colony 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>Colony 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 Thermo Scientific Taq DNA Polymerase (recombinant), 5U/μl</br>
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Protocol
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<table width="580" border="2">
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1. Excise gel slice containing DNA fragment of interest.
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  <tr>
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2. Add 3×sample volume of Buffer DE-A.
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    <td></td>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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    <td>Volume (μl)</td>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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  </tr>
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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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    <td>10×Taq Buffer</td>
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    <td>5</td>
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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  </tr>
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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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    <td>dNTP Mix, 2 mM each</td>
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    <td>5 (0.2 mM of 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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  </tr>
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<tr>
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    <td>Forward primer</td>
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    <td>0.1-1.0 μM</td>
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  </tr>
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<tr>
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    <td>Reverse primer</td>
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    <td>0.1-1.0 μM</td>
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  </tr>
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<tr>
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    <td>25 mM MgCl<sub>2</sub>*</td>
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    <td>1-4 mM</td>
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  </tr>
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<tr>
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    <td>Template DNA</td>
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    <td>Picked from the colony after transformation</td>
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  </tr>
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<tr>
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    <td>Taq DNA Polymerase</td>
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    <td>0.25 U</td>
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  </tr><tr>
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    <td>ddH<sub>2O</sub></td>
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    <td>To 10</td>
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  </tr>
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<tr>
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    <td>Total</td>
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    <td>10</td>
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  </tr>
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</table>
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</br>
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*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:</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>Final concentration of MgCl2(mM)</td>
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    <td>1</td>
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    <td>1.5</td>
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    <td>2</td>
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    <td>2.5</td>
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    <td>3</td>
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    <td>4</td>
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  </tr>
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  <tr>
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    <td>Volume of 25 mM MgCl2 to be added for 10 μl reaction(μl)</td>
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    <td>0.4</td>
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    <td>0.6</td>
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    <td>0.8</td>
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    <td>1.0</td>
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    <td>1.2</td>
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    <td>1.6</td>
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  </tr>
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</table>
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</br>
 +
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>
 +
    <td>Step</td>
 +
    <td>Temperature, °C</td>
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    <td>Time</td>
 +
    <td>Number of cycles</td>
 +
 
 +
  </tr>
 +
  <tr>
 +
    <td>Initial denaturation</td>
 +
    <td>95</td>
 +
    <td>10 min</td>
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    <td>1</td>
 +
 
 +
  </tr>
 +
  <tr>
 +
    <td>Denaturation</td>
 +
    <td>95</td>
 +
    <td>30s</td>
 +
    <td>25~40</td>
 +
 
 +
  </tr>
 +
  <tr>
 +
    <td>Annealing</td>
 +
    <td>Tm-5</td>
 +
    <td>30s</td>
 +
    <td>25~40</td>
 +
 
 +
  </tr>
 +
  <tr>
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    <td>Extension</td>
 +
    <td>72</td>
 +
    <td>1 min/kb</td>
 +
    <td>25~40</td>
 +
 
 +
  </tr>
 +
  <tr>
 +
    <td>Final Extension</td>
 +
    <td>72</td>
 +
    <td>5-15 min</td>
 +
    <td>1</td>
 +
 
 +
  </tr>
   
   
 +
 +
</table>
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 +
</p></div>
</p></div>
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Latest revision as of 08:56, 27 September 2013

Colony PCR

Performed with Thermo Scientific Taq DNA Polymerase (recombinant), 5U/μl

Volume (μl)
10×Taq Buffer 5
dNTP Mix, 2 mM each 5 (0.2 mM of each)
Forward primer 0.1-1.0 μM
Reverse primer 0.1-1.0 μM
25 mM MgCl2* 1-4 mM
Template DNA Picked from the colony after transformation
Taq DNA Polymerase 0.25 U
ddH2O To 10
Total 10

*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:

Final concentration of MgCl2(mM) 1 1.5 2 2.5 3 4
Volume of 25 mM MgCl2 to be added for 10 μl reaction(μl) 0.4 0.6 0.8 1.0 1.2 1.6

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 95 10 min 1
Denaturation 95 30s 25~40
Annealing Tm-5 30s 25~40
Extension 72 1 min/kb 25~40
Final Extension 72 5-15 min 1

Notebook

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