Polymerase Chain Reaction (PCR) protocol

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Solutions/reagents:

• Buffer
• dNTPs
• MgCl ₂
• DMSO
• Upstream primer
• Downstream primer
• Template DNA
• dH₂O
• DNA Polymerase

Equipment:

• Thermocycler
• Reaction tubes

Steps:

1. For Promega GoTaq:
   Set up a reaction in reaction tube (1) as follows on ice:
   
   

   	Initial concentration	Final concentration	Final volume per 50 µl reaction
   Buffer	5X	1X	10 µl
   dNTPs	1.25mM	0.25mM	10 µl
   MgCl 2	25mM	2.5mM	5 µl
   DMSO	50%	5%	5 µl
   Upstream primer	50pmol	1pmol	1 µl
   Downstream primer	50pmol	1pmol	1 µl
   Template DNA	??	~0.5µg	1 µl
   dH2O	N/A	N/A	16.75 µl
   DNA Polymerase	5u/?L	1.25u	0.25 µl

2. For Roche High Fidelity:
   Set up a reaction in reaction tube (1) as follows on ice:
   
   

   	Initial concentration	Final concentration	Final volume per 50 µl reaction
   Buffer	10X	1X	5 µl
   dNTPs	1.25mM	0.25mM	10 µl
   DMSO	50%	5%	5 µl
   Upstream primer	50pmol	1pmol	1 µl
   Downstream primer	50pmol	1pmol	1 µl
   Template DNA	??	~0.5µg	1 µl
   dH2O	N/A	N/A	16.75 µl
   DNA Polymerase	5u/?L	2.5u	0.5 µl

3. An example program:
   Program a standard thermocycler to run the reaction using the following parameters:
   Initial denaturation
   
   • Denature: 96°C, 5 mins
   This denatures any double stranded DNA.
   Thermocycling
   
   • No. of cycles: 25
   • Denature: 96°C, 1 min
   • Anneal: 55°C, 30 secs
   • Elongate: 72°C, 1 min
   Termination
   • Elongate: 72°C, 5 mins
   • Hold: 4°C, until removed from machine
   Final extension for incomplete strands and holding of sample at low temperature.
   

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 15 mins