Team:Calgary/Notebook/Protocols/Process9

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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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Revision as of 03:00, 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 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


Follow the same protocol for a colony PCR.

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