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