Team:Groningen/protocols/PCR

From 2013.igem.org

(Difference between revisions)
Line 49: Line 49:
     <td><ul>MQ water</td>
     <td><ul>MQ water</td>
     <td><ul>up to 50 &micro;l</td>
     <td><ul>up to 50 &micro;l</td>
-
     <td><dd></td>
+
     <td><ul></td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><dd>5x Phusion Buffer</td>
+
     <td><ul>5x Phusion Buffer</td>
-
     <td><dd>10 &micro;l</td>
+
     <td><ul>10 &micro;l</td>
-
     <td>1x</td>
+
     <td><ul>1x</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>DMSO 5%</td>
+
     <td><ul>DMSO 5%</td>
-
     <td>1.5 &micro;l</td>
+
     <td><ul>1.5 &micro;l</td>
-
     <td></td>
+
     <td><ul></td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>10 mM dNTPs</td>
+
     <td><ul>10 mM dNTPs</td>
-
     <td>1&micro;l</td>
+
     <td><ul>1&micro;l</td>
-
     <td>200 &micro;M</td>
+
     <td><ul>200 &micro;M</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>primer F</td>
+
     <td><ul>primer F</td>
-
     <td>2.5 &micro;l</td>
+
     <td><ul>2.5 &micro;l</td>
-
     <td>0.5 &micro;M</td>
+
     <td><ul>0.5 &micro;M</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>primer R</td>
+
     <td><ul>primer R</td>
-
     <td>2.5 &micro;l</td>
+
     <td><ul>2.5 &micro;l</td>
-
     <td>0.5 &micro;M</td>
+
     <td><ul>0.5 &micro;M</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>template DNA</td>
+
     <td><ul>template DNA</td>
-
     <td>1 &micro;l</td>
+
     <td><ul>1 &micro;l</td>
-
     <td>&sim;1 ng</td>
+
     <td><ul>&sim;1 ng</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>Phusion Polymerase <br>2 U/&micro;l</td>
+
     <td><ul>Phusion Polymerase <br>2 U/&micro;l</td>
-
     <td>0.3 &micro;l</td>
+
     <td><ul>0.3 &micro;l</td>
-
     <td>0.01 U/&micro;l</td>
+
     <td><ul>0.01 U/&micro;l</td>
   </tr>
   </tr>

Revision as of 14:38, 12 September 2013

PCR

Materials:
  • PCR tubes
  • MQ water
  • DMSO 5%
  • Phusion buffer
  • DNTPs
  • F- and R-primer
  • Phusion Polymerase
  • DNA template


PCR reaction mixture:
Component 50 µl Final concentration
    MQ water
    up to 50 µl
      5x Phusion Buffer
      10 µl
      1x
      DMSO 5%
      1.5 µl
        10 mM dNTPs
        1µl
        200 µM
        primer F
        2.5 µl
        0.5 µM
        primer R
        2.5 µl
        0.5 µM
        template DNA
        1 µl
        ∼1 ng
        Phusion Polymerase
        2 U/µl
        0.3 µl
        0.01 U/µl


      Cycling instructions:
      Temperature Time Number of Cycles
      98°C 2 min 1
      98°C 10 sec 34
      Tm-5°C 25 sec
      72°C 30 sec/kb
      72°C 10 min 1
      4°C ∞ 1

      Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/f-530s-product-information.pdf