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Polymerase Chain Reaction (PCR) to Amplify rRNA Gene Fragment

2024-11-13 PCR 加入收藏

Polymerase Chain Reaction (PCR) to Amplify rRNA Gene Fragment Prepare sufficient

Polymerase Chain Reaction (PCR) to Amplify rRNA Gene Fragment Prepare 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

Notes:

1 To improve specificity, template DNA concentration, annealing temperature and Mg2+ concentration may be varied.

2 1 minute extension time should be used for each kbp of product expected.

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