PrbbBB:colony pcr v1

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(Materials)
(Procedure)
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==Procedure==
==Procedure==
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'''II PCR reaction'''
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'''I PCR reaction'''
<table>
<table>
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   <table frame=box>
   <table frame=box>
   <tr align=right>
   <tr align=right>
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   <td></td> <th width=100>100µl single reaction</th> <th width=100>3.5xMaster</th> <th width=100>10xMaster</th></tr>
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   <td></td> <th width=100>11µl single reaction</th> <th width=100>60xMaster</th> <th width=100>110xMaster</th></tr>
    
    
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   <tr align=right> <td>H2O</td>               <td>76µl</td>    <td>266</td>    <td>760</td> </tr>
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   <tr align=right> <td>H2O</td>                 <td>7.5µl</td>    <td></td>    <td></td> </tr>
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   <tr align=right> <td>5x HF Buffer</td>     <td>20µl</td>    <td>70</td>     <td>200</td> </tr>
+
   <tr align=right> <td>10x Amplitaq buffer</td> <td>1.1µl</td>    <td></td>   <td></td> </tr>
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   <tr align=right> <td>10mM dNTP</td>         <td>2µl</td>     <td>7</td>     <td>20</td> </tr>
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   <tr align=right> <td>10mM dNTP</td>           <td>0.22µl</td>   <td></td>   <td></td> </tr>
   <tr><td></td></tr>
   <tr><td></td></tr>
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   <tr align=right> <td>FW primer 100 µM</td>    <td>0.5µl</td>   <td>1.75</td>   <td>5</td> </tr>
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   <tr align=right> <td>VF2 primer 10 µM</td>    <td>0.55µl</td>   <td></td>   <td></td> </tr>
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   <tr align=right> <td>RV primer 100 µM</td>    <td>0.5µl</td>   <td>1.75</td>   <td>5</td> </tr>
+
   <tr align=right> <td>VR  primer 10 µM</td>    <td>0.55µl</td>   <td></td>   <td></td> </tr>
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   <tr align=right> <td>Phusion</td>           <td>1µl</td>     <td>3.5</td>    <td>10</td> </tr>
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   <tr align=right> <td>AmpliTaq 5U/µl</td>       <td>0.1µl</td>   <td></td>    <td></td> </tr>
   <tr><td></td></tr>
   <tr><td></td></tr>
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   <tr align=right> <td>template DNA</td>     <td>0.1µl</td>   <td>--</td>   <td>--</td> </tr>
+
   <tr align=right> <td>template DNA</td>         <td>1µl</td>     <td>--</td> <td>--</td> </tr>
   </table>
   </table>
</td>
</td>

Revision as of 07:55, 26 February 2009

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Contents

Overview

Materials

Procedure

I PCR reaction

11µl single reaction 60xMaster 110xMaster
H2O 7.5µl
10x Amplitaq buffer 1.1µl
10mM dNTP 0.22µl
VF2 primer 10 µM 0.55µl
VR primer 10 µM 0.55µl
AmpliTaq 5U/µl 0.1µl
template DNA 1µl -- --
PCR Program
30"@98°C
5x (10"@98°C; 15"@Ta; text@72°C);
25x (10"@98°C; text@72°C);
10'@72°C
∞ 4°C
  • extension time text = (kb insert length) × 25"
  • annealing temperature Ta = (primer annealing) + 3°C

II Agarose Gel

Notes

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

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References

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