Team:Calgary/Sandbox/Notebook/Protocols/KapaPCR

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<li>Add the mix to each tube</li>
<li>Add the mix to each tube</li>
<li>Set the following program in the thermocycler:</li>
<li>Set the following program in the thermocycler:</li>
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<ul>
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<ol>
<li>Stage 1 (1 cycle): 95°C for 2 to 5 min</li>
<li>Stage 1 (1 cycle): 95°C for 2 to 5 min</li>
<li>Stage 2 (30 cycles): 98°C for 20 sec, 55°C for 15 sec, 72°C for 1min/kb</li>
<li>Stage 2 (30 cycles): 98°C for 20 sec, 55°C for 15 sec, 72°C for 1min/kb</li>
<li>Stage 3 (1 cycle): 72°C for 1 to 5 min</li>
<li>Stage 3 (1 cycle): 72°C for 1 to 5 min</li>
<li>Hold at 4°C</li>
<li>Hold at 4°C</li>
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</ul>
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</ol>
<li>Run the products in a agarose gel</li>
<li>Run the products in a agarose gel</li>
</ol>
</ol>

Latest revision as of 03:42, 19 September 2013

Kapa PCR (for DNA constructions)

Mix (per reaction)

  • 5.0µL 5x Kapa Hifi Buffer
  • 0.75µL 10mM dNTPs
  • 0.75µL Primer Forward
  • 0.75µL Primer Reverse
  • 0.5µL Kapa Hifi DNA polymerase
  • 10 to 50ng of DNA
  • ddH2O up to 25µL

Protocol

  1. Add the mix to each tube
  2. Set the following program in the thermocycler:
    1. Stage 1 (1 cycle): 95°C for 2 to 5 min
    2. Stage 2 (30 cycles): 98°C for 20 sec, 55°C for 15 sec, 72°C for 1min/kb
    3. Stage 3 (1 cycle): 72°C for 1 to 5 min
    4. Hold at 4°C
  3. Run the products in a agarose gel