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Linear template- TetR

Two step extension PCR

Gene PCR

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
• 0.5 µl Polymerase
• 36.5 µl H2O

Primers: TetR Forward: CTC GAG AAT TCG CCA CCA TGT CCA GAT TAG ATA AAA GTA AAG ; Tm=62.6ºC

TetR-His6 Reverse: GTA GCA GCC TGA GTC GTT ATT AAT GAT GAT GAT GAT GAT GAG AAC CCC CAG ACC CAC TTT CAC ATT TAA G ; Tm=68.5 ºC

PCR Cycle (30 cycles)

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)

5. 72°C for 5 min
6. 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

Extension PCR

Materials

• Gene PCR product
• Extension primers 500nM

PCR Cycle (30 cycles)

1. 98°C for 30s
2. 98°C for 7.5 seconds
3. 58°C for 20 sec 
4. 72°C for 15 sec; 
(calculate for the longest fragment) 
5. 72°C for 5 min
6. 4°C "forever"

Materials

• 1µl Final primers

PCR Cycle (10 cycles)

1. 98°C for 30s
2. 98°C for 7.5 seconds
3. 58°C for 20 sec
4. 72°C for 15 sec;  5. 72°C for 5 min
6. 4°C "forever"