Team:TU Delft/protocols/PCR

From 2010.igem.org

PCR

Materials:

- Pfx Polymerase (Invitrogen)

- 10x Pfx Bufer (Invitrogen)

- Enhancer (Invitrogen)

- 50 mM MgSO4

- 10 mM dNTPs

- Primer solutions 5 pmol/μL

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


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 a 100 μL 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
Primer 1 3 μL
Primer 2 3 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

5. Place samples from the ice in a thermal cycler preheated to 95 °C and start the previously programmed PCR.

PCR program:

Step Annealing Temperature Time, min:sec Number of cycles
Initial denaturation x °C * 2:00 1
Annealing 95 °C 2:00 1
Extension 68 °C 1:00 1
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


* Calculate the optimal annealing temperature = 3x G/C + 2x A/T