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.
