Template:Team:Groningen/CONTENT/Protocols and Protocols/PCR

PCR

Polymerase chain reaction (PCR) is used to amplify DNA.

ML-I

PCR mix

Create the following mix.

Ingredient

20 μL reaction

Final Concentration

\( \mathrm{H_2O}\)

Add to 20 μL

5x Phusion HF buffer*

4 μL

1x

10 mM dNTPs

0.4 μL

200 μM each

Forward primer**

X μL

0.5 μM

Template DNA

X μL

(DMSO***, optional)

(0.6 μL)

(3%)

Phusion DNA polymerase

0.2 μL

0.02 U/ μL

PCR Mix.

Vortex

Gently vortex the samples and spin down.

Thermal Cycle

Place the reactions in a thermal cycler. Perform PCR using recommended thermal cycling conditions.

Cycle Step

Temperature

Time

Cycle

Initial denaturation

98 °C

30 s

1

Denaturation

98 °C

5-10 s

25-35

Annealing

T m - 5°C

10-30 s

25-35

Extension

72 °C

15-30 s/kb

25-35

Final extension

72 °C

5-10 min

1

Hold

4 °C

Hold

1

Thermal Cycling.