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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 H2O
- 0.4 mL TaKaRa Ex-Taq DNA Polymerase (2 units)()
- 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 H2O 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. @ 94oC
- Primer annealing 45 sec. @ 55oC
- Primer extension2 1 min. @ 72oC
Phase 2 - 35 cycles - standard denaturation 1 min. @ 94oC
- Primer annealing 45 sec. @ 55oC
- Primer extension2 1 min. @ 72oC
Phase 3 - 1 cycle - standard denaturation 1 min. @ 94oC
- Primer annealing 45 sec. @ 55oC
- Primer extension2 10 min. @ 72oC
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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