Team:Goettingen/protocol PCR

From 2014.igem.org

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         PCR cycles:</p>
         PCR cycles:</p>
       <table width="500" align="center">
       <table width="500" align="center">
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       <tr><td><b>Amount of cycles </b></td><td> Reaction </td><td> Temp. </td><td> Time </td></tr>
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       <tr><td><b>Amount of cycles </b></td><td><b> Reaction </b></td><td><b> Temp. </b></td><td> <b>Time</b> </td></tr>
       <tr><td>1</td><td>Pre-running</td><td>98°C</td><td>1 min</td></tr>
       <tr><td>1</td><td>Pre-running</td><td>98°C</td><td>1 min</td></tr>
       <tr><td>10</td><td>Denaturation</td><td>98°C</td><td>15 sec</td></tr>
       <tr><td>10</td><td>Denaturation</td><td>98°C</td><td>15 sec</td></tr>

Revision as of 10:51, 2 October 2014

Fusion PCR

Reaction protocol 50 µl:
      4 μl Primer forward (5 pmol)
      4 μl Primer reverse (5 pmol)
      100 ng template-DNA of each fragment
      10 μl 5x HF-Buffer
      1 μl PfuS-Polymerase
      2 μl dNTPs (12.5 mM)
      ad. 50 μl sterile water

PCR cycles:

Amount of cycles Reaction Temp. Time
1Pre-running98°C1 min
10Denaturation98°C15 sec
10Annealing52°C30 sec
10Primer Extension 72°C2 min
1Pause8°Cadd primer
21Denaturation98°C15 sec
21 Annealing52°C30 sec
21Primer Extension72°C2 min + 5 sec/cycle
1Final elongation72°C10 min
1 Hold 4°C

Pfus PCR

Watch the following tutorial to learn about the whole process:



Reaction protocol for 50 µl
      1 μl Primer fwd (5 pmol)
      1 μl Primer rev (5 pmol)
      1 μl Matrizen-DNA (ca. 100 ng)
      10 μl 5x HF-Polymerase Puffer
      0.5 μl PfuS-Polymerase (50 ng)
      1 μl dNTPs (12.5 μmol ml-1)
      35.5 μl dest. Water

PCR cycles:

CyclesReactionTemp.Time
1Pre-running98 °C10 min
30Denature9810 s
30Annealing54-60 °C1 min
30Primer Extension72 °C15 s - 2.5 min
1Final elongation72 °C10 min
1Hold4 °C


Gradient PCR