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From 2010.igem.org

Overlapping PCR

Materials:

- Pfx polymerase (Invitrogen)

- 10x Pfx Buffer (Invitrogen)

- enhancer (Invitrogen)

- 50 mM MgSO4 (Invitrogen)

- 10 mM dNTPs

- primer solutions 5 mol/mL

- template DNA (plasmid at 50 pg – 1 ng/μL)

- PCR machine

Protocol:

First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw them on ice. Preparation of reaction mixture:

1. Gently vortex and briefly centrifuge all solutions after thawing

2. Keep solutions on ice

3. Add to a thin walled PCR tube, on ice, for PCR reaction:

1× pre-mix

Component	Sample
Pfx polymerse	0.6 μL
10x Pfx buffer	5 μL
Enhancer	5 μL
10 mM dNTPs	1.5 μL
50 mM MgSO4	1 μL
DNA template	1 μL
H20	29.9 μL

4. Gently vortex the sample and briefly centrifuge (5 sec) to collect all droplets at the bottom of the tube

PCR program:

Step	Annealing Temperature	Time, min:sec	Number of cycles
Initial denaturation	95 °C	2:00	1
Annealing	X °C *	1:00	1
Extension	68 °C	1:00	1
Denaturation	95 °C	1:00	5
Annealing	x °C *	1:00	5
Extension	68 °C	1:00	5
Add primers (3 μL primer 1 + 3 μL primer 2)
Denaturation	95 °C	1:00	25
Annealing	x °C *	1:00	25
Extension	68 °C	1:00	25
Final Extension	68 °C	10:00	1

* Annealing temperature dependent on primer. Optimal temperature: 3x G/C + 2x A/T