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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

• 10µl 5x iProof HF buffer
• 0.5 µl of each extension primer@500nM
• 1 µl dNTP mix of 10mM
• 0.5 µl High Fidelity Polymerase
• 1 µl of gene specific PCR product from previous step
• 0.5 µl Polymerase
• 36.5 µl H2O

Extension Primers:

Forward: GAT CTT AAG GCT AGA GTA CTA ATA CGA CTC ACT ATA GGG AAT ACA AGC TAC TTG TTC TTT TTG CAC TCG AGA ATT CGC CAC C Tm=68.4 ºC

Reverse: CAA AAA ACC CCT CAA GAC CCG TTT AGA GGC CCC AAG GGG TTA TGC TAG TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GTA GCA GCC TGA GTC G; Tm=70.4ºC

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"

Materials

• 10µl 5x iProof HF buffer
• 0.5 µl of each final primer @50µM/1µl of primer mix@1µM
• 1 µl dNTP mix of 10mM
• 0.5 µl High Fidelity Polymerase
• 1 µl of previous PCR product(extension PCR)
• 0.5 µl Polymerase
• 36.5 µl H2O

Final primers

Forward: GAT CTT AAG GCT AGA GTA C; Tm=46.0ºC

Reverse: CAA AAA ACC CCT CAA GAC; Tm=48.6ºC

PCR Cycle (30 cycles)

1. 98°C for 30s
2. 98°C for 7.5 seconds
3. 53°C for 20 sec
4. 72°C for 15 sec; 
5. 72°C for 5 min
6. 4°C "forever"