PolymeraseChainReaction(PCR)toAmplifyrRNAGeneFragment

1. 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)(Panvera)

• 90 mL
  
  

2. Aliquot 45 mL of master mix into each of two 0.5 mL microfuge tubes labelled with the student's initials; PCR; date.
   
   

3. Add 1-5 mL template DNA¹ (100 ng for bacterial genomic DNA), 0-4 mL H₂O to yield a final volume of 50 mL.
   
   

4. Add 50 mL mineral oil (using cut pipette tip), close tube tightly, place in thermal cycler.
   
   

5. Initiate thermal cycling program.

PCR55

Phase 1 - 1 cycle

• Initial denaturation 4 min. @ 94^oC

• Primer annealing 45 sec. @ 55^oC

• Primer extension² 1 min. @ 72^oC

Phase 2 - 35 cycles

• standard denaturation 1 min. @ 94^oC

• Primer annealing 45 sec. @ 55^oC

• Primer extension² 1 min. @ 72^oC

Phase 3 - 1 cycle

• standard denaturation 1 min. @ 94^oC

• Primer annealing 45 sec. @ 55^oC

• Primer extension² 10 min. @ 72^oC

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.