Polymerase Chain Reaction (PCR) to Amplify rRNA Gene Fragment

Polymerase Chain Reaction (PCR) to Amplify rRNA Gene FragmentPrepare sufficient master mix for both partners (45 mL/50 mL reaction)

10 mL 10x PCR buffer
10 mL 2.5 mM dNTPs (0.25 mM final concentration)
15 mL Primer A (5 pmole/mL)
15 mL Primer B (5 pmole/mL)
40 mL H2 O
0.4 mL TaKaRa Ex-Taq DNA Polymerase (2 units)( Panvera )
90 mL

Aliquot 45 mL of master mix into each of two 0.5 mL microfuge tubes labelled with the student's initials; PCR; date. Add 1-5 mL template DNA1 (100 ng for bacterial genomic DNA), 0-4 mL H2 O to yield a final volume of 50 mL. Add 50 mL mineral oil (using cut pipette tip), close tube tightly, place in thermal cycler. Initiate thermal cycling program.

PCR55

Phase 1 - 1 cycle
- Initial denaturation 4 min. @ 94o C
- Primer annealing 45 sec. @ 55o C
- Primer extension2 1 min. @ 72o C
Phase 2 - 35 cycles
- standard denaturation 1 min. @ 94o C
- Primer annealing 45 sec. @ 55o C
- Primer extension2 1 min. @ 72o C
Phase 3 - 1 cycle
- standard denaturation 1 min. @ 94o C
- Primer annealing 45 sec. @ 55o C
- Primer extension2 10 min. @ 72o C