Polymerase Chain Reaction (PCR) to Amplify rRNA Gene Fragment

 

<center> <b><font>Polymerase Chain Reaction (PCR) to Amplify rRNA Gene Fragment</font> </b></center>

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 H₂ O
   • 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 H₂ O 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^o C
  • Primer annealing 45 sec. @ 55^o C
  • Primer extension² 1 min. @ 72^o C
Phase 2 - 35 cycles
• • standard denaturation 1 min. @ 94^o C
  • Primer annealing 45 sec. @ 55^o C
  • Primer extension² 1 min. @ 72^o C
Phase 3 - 1 cycle
• • standard denaturation 1 min. @ 94^o C
  • Primer annealing 45 sec. @ 55^o C
  • Primer extension² 10 min. @ 72^o 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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