Team:Groningen/Protocols and Protocols/PCR
Blue Bio Energy
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.