Difference between revisions of "Team:UiOslo Norway/Experiments/PCR phusion"

Line 39: Line 39:
 
   <tr>
 
   <tr>
 
     <td>Initial Denaturation</td>
 
     <td>Initial Denaturation</td>
−
     <td>95 °C</td>
+
     <td>98 °C</td>
−
     <td>30 seconds /5 Minutes for Colony PCR
+
     <td>30 seconds</td>
−
  </td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
 
     <td rowspan="3"></br></br>35 Cycles</td>
 
     <td rowspan="3"></br></br>35 Cycles</td>
−
     <td>95 °C</td>
+
     <td>98 °C</td>
−
     <td>30 seconds</td>
+
     <td>10 seconds</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
     <td>45 °C – 72 °C</td>
 
     <td>45 °C – 72 °C</td>
−
     <td>60 seconds</td>
+
     <td>30 seconds</td>
 
   </tr>
 
   </tr>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
−
     <td>68 °C</td>
+
     <td>72 °C</td>
−
     <td>1 minute / 1 kb</td>
+
     <td>30 seconds / 1 kb</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
 
     <td>Final Extension</td>
 
     <td>Final Extension</td>
−
     <td>68 °C</td>
+
     <td>72 °C</td>
 
     <td>5 minutes</td>
 
     <td>5 minutes</td>
 
   </tr>
 
   </tr>

Revision as of 10:13, 14 August 2015

PCR with Phusion-HF DNA Polymerase (NEB):

Back to Protocols


  1. Reaction Setup:

    10 µl 5X Phusion HF Buffer
    5 µl dNTPS [2 mM]
    2.5 µl Forward Primer [10 µM]
    2.5 µl Reverse Primer [10 µM]
    1 µl Template DNA
    0.5 µl Phusion HF DNA Polymerase
    28.5 µl MilliQ Water

    50 µl Total Reaction


  2. PCR Program:

    Step Temperature Time
    Initial Denaturation 98 °C 30 seconds


    35 Cycles    		 98 °C 10 seconds
    45 °C – 72 °C 30 seconds
    72 °C 30 seconds / 1 kb
    Final Extension 72 °C 5 minutes
    Hold 4 °C forever