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Linear template- TetR

Two step extension PCR

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Materials

• 10µl 5x iProof HF buffer
• 0.5 µl of each primer
• 1 µl dNTP mix of 10mM
• 0.5 µl High Fidelity Polymerase
• 1 µl DNA template
• 36.5 µl H2O

Protocol

• Put mix in PCR thermal cycler
• Program the PCR thermal cycler: 1. 98°C for 30s 2. 98°C for 7.5 seconds 3. 58°C for 20 sec (this depends on the primers, therefore look up the annealing temperature of the primers used) 4. 72°C for 15 sec; you have to calculate the time required. This polymerase has an effciency of 15-30s/kb. (calculate for the longest fragment) 6. 72°C for 5 min 7. 4°C "forever"
• Save the file in the MAIN folder→ OK→ SAVE→RUN→Select the block you use→ RUN→ Change sample volume to 50µl, LidT should be 105°C and the box just below ("hotlid") should ALWAYS be checked
• Press STATUS to check the progress