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Revision as of 21:17, 21 August 2015
PCR mix
1. 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.
2. Gently vortex the samples and spin down.
3. 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.