PrbbBB:colony pcr v1: Difference between revisions

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   <table frame=box>
   <table frame=box>
   <tr><th></th>        <th>PCR Program</th>
   <tr><th></th>        <th>PCR Program</th>
  <tr><td></td>  <td> 10' @95°C  </td></tr>


   <tr><td></td>   <td> 30"@98°C  </td></tr>
   <tr><td>30x</td> <td> (30" @98°C; 15" @64; ''t<sub>ext</sub>''@72°C); </td></tr>
  <tr><td>5x</td> <td> (10"@98°C; 15"@''T<sub>a</sub>''; ''t<sub>ext</sub>''@72°C); </td></tr>
   <tr><td></td>   <td> optional: 10' @72°C  </td></tr>
  <tr><td>25x</td><td> (10"@98°C; ''t<sub>ext</sub>''@72°C); </td></tr>
   <tr><td></td>   <td> ∞ 4°C  </td></tr>
   <tr><td></td>   <td> 10'@72°C  </td></tr>
   <tr><td></td>   <td> ∞ 4°C  </td></tr>
   </table>
   </table>


* extension time '''t<sub>ext</sub>''' = (kb insert length) × 25"
* extension time '''t<sub>ext</sub>''' = (kb insert length) × 1' (1 min)
* annealing temperature '''T<sub>a</sub>''' = (primer annealing) + 3°C
    
    
</td><tr>
</td><tr>

Revision as of 05:03, 26 February 2009

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Overview

Materials

Procedure

I PCR reaction

11µl single reaction 60xMaster 115xMaster
H2O 7.5µl 450 862
10x Amplitaq buffer 1.1µl 66 126.5
10mM dNTP 0.22µl 13.2 25.3
VF2 primer 10 µM 0.55µl 33 63.3
VR primer 10 µM 0.55µl 33 63/3
AmpliTaq 5U/µl 0.1µl 6 11.5
template DNA 1µl -- --
PCR Program
10' @95°C
30x (30" @98°C; 15" @64; text@72°C);
optional: 10' @72°C
∞ 4°C
  • extension time text = (kb insert length) × 1' (1 min)

II Agarose Gel

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

raik: no comment


References

Contact

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