Team:Calgary/Notebook/Protocols/Process9

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Plasmid Extraction

Taq PCR Protocol 

     <td>540 μL</td>
   </tr>
   <tr>
     <td>10X Taq Buffer</td>
     <td>5 μL</td>
     <td>15 μL</td>
     <td>25 μL</td>
     <td>75 μL</td>
   </tr>
   <tr>
     <td>2mM dNTPs</td>
     <td>5 μL</td>
     <td>15 μL</td>
     <td>25 μL</td>
     <td>75 μL</td>
   </tr>
   <tr>
     <td>Forward Primer (100 ug/ul) </td>
     <td>1 μL</td>
     <td>3 μL</td>
     <td>5 μL</td>
     <td>15 μL</td>
   </tr>
   <tr>
     <td>Reverse Primer (100 ug/ul) </td>
     <td>1 μL</td>
     <td>3 μL</td>
     <td>5 μL</td>
     <td>15 μL</td>
   </tr>
   <tr>
     <td>50mM MgCl2</td>
     <td>1.5 μL</td>
     <td>4.5 μL</td>
     <td>7.5 μL</td>
     <td>22.5 μL</td>
   </tr>
   <tr>
     <td>Taq Polymerase (50 ug/ul)</td>
     <td>0.5 μL</td>
     <td>1.5 μL</td>
     <td>2.5 μL</td>
     <td>7.5 μL</td>
   </tr>
 </table>

Thermocycler Conditions

  1. 1 Cycle - 6 minutes at 95 degrees Celsius
  2. 36 cycles of:
    1. 1 minute at 95 degrees Celsius
    2. 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content )
    3. 1 minute at 72 degrees Celsius
  3. 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius

Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 or even 3 minutes

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Retrieved from "http://2011.igem.org/Team:Calgary/Notebook/Protocols/Process9"
Reagent Volume ( 1x ) Volume ( 3x ) Volume ( 5x ) Volume ( 15x )
Sterile H2O 36 μL 108 μL 180 μL