Template:Team:Groningen/CONTENT/PROTOCOLS/PCR

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.

Restriction

Component

Plasmid DNA

Unpurified Product

Genomic DNA

Water, nuclease free*

up to 20 μl

up to 20 μl

up to 50 μl

10x NEB 2.1 buffer

2 μl

2 μl**

5 μl

DNA

up to 1 μg

up to 0.2 μg

up to 5 μg

NEB Enzyme

0.5 μl

0.5 μl

0.5 μl

Total Volume

20 μl

20 μl

20 μl

Restriction.