Team:Calgary/Notebook/Protocols/Process9

From 2011.igem.org

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<p>The following is obtained from the Invitrogen Taq PCR kit (although most PCR kits have similar protocols).</p>
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   <p><b> Thermocycler Conditions </b></p>
   <p><b> Thermocycler Conditions </b></p>
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     <li> 1 Cycle - 6 minutes at 95 degrees Celsius </li>
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     <li> 1 Cycle - 6 minutes at 95&deg;C </li>
     <li> 36 cycles of:
     <li> 36 cycles of:
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         <li> 1 minute at 95 degrees Celsius </li>
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         <li type="a"> 1 minute at 95&deg;C </li>
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         <li> 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content ) </li>
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         <li type="a"> 1 minute at 58&deg;C (this step done at 65&deg;C for higher GC content ) </li>
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         <li> 1 minute at 72 degrees Celsius </li>
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         <li type="a"> 1 minute at 72&deg;C </li>
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     <li> 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius </li>
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     <li> 1 Cycle - 10 minutes at 72&deg;C then HOLD at 4&deg;C</li>
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   <p> 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</p>
   <p> 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</p>
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<p>Follow the same protocol for a colony PCR.</p>
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Latest revision as of 04:26, 29 September 2011


Taq PCR Protocol

The following is obtained from the Invitrogen Taq PCR kit (although most PCR kits have similar protocols).

Taq PCR Protocol

Reagent Volume ( 1x ) Volume ( 3x ) Volume ( 5x ) Volume ( 15x )
Sterile H2O 36 μL 108 μL 180 μL 540 μL
10X Taq Buffer 5 μL 15 μL 25 μL 75 μL
2mM dNTPs 5 μL 15 μL 25 μL 75 μL
Forward Primer (100 ug/ul) 1 μL 3 μL 5 μL 15 μL
Reverse Primer (100 ug/ul) 1 μL 3 μL 5 μL 15 μL
50mM MgCl2 1.5 μL 4.5 μL 7.5 μL 22.5 μL
Taq Polymerase (50 ug/ul) 0.5 μL 1.5 μL 2.5 μL 7.5 μL

Thermocycler Conditions

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

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


Follow the same protocol for a colony PCR.

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