Team:USTC CHINA/Notebook/Protocols/Colony PCR

From 2013.igem.org

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*Volumes of 25 mM MgCl2, required for specific final MgCl2 concentration:</br>
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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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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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<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>95</td>
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    <td>10 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>95</td>
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    <td>30s</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>Tm-5</td>
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    <td>30s</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>72</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>72</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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</p></div>
</p></div>

Revision as of 16:52, 25 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"